Small molecule RNase inhibitors and methods of use

ABSTRACT

Small molecule inhibitors of bacterial ribonuclease (e.g., RnpA) and methods for their synthesis and use are described herein. The methods of using the compounds include treating and preventing microbial infections and inhibiting bacterial ribonuclease. Also described herein are methods of identifying compounds for treating or preventing a microbial infection.

CROSS-REFERENCE TO PRIORITY APPLICATIONS

This application claims priority to U.S. Provisional Application No.61/436,342, filed Jan. 26, 2011, which is incorporated herein byreference in its entirety.

STATEMENT REGARDING FEDERALLY FUNDED RESEARCH

This invention was made with government support under grant numberAI073780 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD

The subject matter disclosed herein generally relates to small moleculeinhibitors of bacterial ribonuclease (RNase) and methods of theirpreparation. Also, the subject matter described herein generally relatesto methods of using the small molecule inhibitors described herein totreat and prevent microbial infections.

BACKGROUND

Staphylococcus aureus infections are often associated with high rates ofmorbidity and mortality (see Shorr et al., Crit Care Med, 34: 2588-2595(2006)). Indeed, reports estimate that in 2005 the organism caused moreU.S. deaths than HIV/AIDS (see Bancroft, E. A., Jama, 298: 1803-1804(2007); Klevens et al., Jama, 298: 1763-1771 (2007)). The emergence ofvancomycin-resistant, methicillin-resistant, multidrug-resistant, andhypervirulent strains has further accentuated the need for novelantibiotics (see Appelbaum, P. C., Int J Antimicrob Agents, 30: 398-408(2007); Zetola et al., Lancet Infect Dis, 5: 275-286 (2005)). BacterialRNA processing and degradation are required cellular processes that canbe exploited for antimicrobial drug discovery.

Much of the understanding of bacterial RNA degradation comes fromstudies of Escherichia coli where bulk mRNA decay is thought to becatalyzed by a holoenzyme complex (RNA degradosome), which consists ofat least four subunits: RNase E (me), RNA helicase (rhlB), enolase(eno), and PNPase (pnpA) (see Carpousis, A. J., Annu Rev Microbiol, 61:71-87 (2007)). RNase E is an essential ribonuclease and a key componentof the degradosome complex. It serves as a scaffold for the assembly ofother members of the RNA degradosome and catalyzes the initialendoribonucleolytic event during substrate degradation (see Mackie, G.A., Nature, 395: 720-723 (1998); Vanzo et al., Genes Dev, 12: 2770-2781(1998)). Based on its essentiality, RNase E could be considered anappropriate target for antibiotic drug discovery. However, manyGram-positive bacteria, including S. aureus, lack an RNase E amino acidortholog (see Condon, C., Microbiol Mol Biol Rev, 67: 157-174 (2003)).As a consequence, their degradation components and mechanism(s) of mRNAdecay are less understood.

Recent studies suggest that at least two ribonucleases, RNase J1 andRNase Y, contribute to bulk mRNA degradation within Bacillus subtilis,and presumably other Gram-positive bacteria. B. subtilis ribonuclease J1is a bifunctional ribonuclease, with 5′ exonuclease and endonucleaseactivities, that mediates mRNA degradation in vitro (see Even et al.,Nucleic Acids Res, 33: 2141-2152 (2005); Mathy et al., Cell, 129:681-692 (2007)). The enzyme has also been found to interact with enolase(a component of the E. coli RNA degradosome) and RNase J1 depleted B.subtilis strains demonstrate a moderate decrease in mRNA decay,suggesting that it may be the functional equivalent to E. coli RNase E(see Even et al., Nucleic Acids Res, 33: 2141-2152 (2005); Commichau etal., Mol Cell Proteomics, 8: 1350-1360 (2009); Mader et al., MolMicrobiol, 70: 183-196 (2008)). However, mRNA turnover still occurs inRNase J1 diminished cells and RNA species containing 5′ strong-hairpinstructures are not effectively degraded by the enzyme, indicating thatadditional factors are likely to contribute to B. subtilis cellular RNAdegradation (see Yao et al., Rna, 15: 2331-2339 (2009)). Ribonuclease Yis a recently identified endonuclease that may ostensibly work inconcert with RNase J1 to mediate bulk RNA decay. RNase Y can cleave mRNAmolecules containing high-order secondary structures and globallyaffects cellular messenger RNA turnover (see Shahbabian et al., Embo J,28: 3523-3533 (2009)). Both RNase J1 and RNase Y are essential enzymesand, in that regard, could be considered targets for antimicrobial drugdiscovery (see Kobayashi et al., Proc Natl Acad Sci USA, 100: 4678-4683(2003)). However, it remains to be seen whether RNase J1, RNase Y,and/or previously uncharacterized ribonucleases modulate mRNA decaywithin S. aureus.

SUMMARY

In accordance with the purposes of the disclosed materials, compounds,compositions, kits, and methods, as embodied and broadly describedherein, the disclosed subject matter relates to compositions, methods ofmaking said compositions, and methods of using said compositions. Morespecifically, compounds and compositions for use as inhibitors ofbacterial ribonuclease (RNase) are provided herein. A class of RNaseinhibitors includes compounds of the following structure:

and pharmaceutically acceptable salts and prodrugs thereof. In thesecompounds,

is a single or double bond; A¹, A², A³, A⁴, and A⁵, are eachindependently selected from N or CR¹; A⁶, A², A⁸, A⁹, and A¹⁰ are eachindependently selected from N or CR²; each R¹, each R², R³, R⁵, R⁶, R²,and R⁸ are independently selected from hydrogen, halogen, hydroxyl,cyano, nitro, substituted or unsubstituted amino, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedheteroalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted heteroalkynyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedalkoxyl, substituted or unsubstituted aryloxyl, or substituted orunsubstituted carboxyl; and R⁴ is hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted heteroalkenyl,substituted or unsubstituted alkynyl, or substituted or unsubstitutedheteroalkyl. R⁶ and R⁷ can optionally combine to form substituted orunsubstituted aryl or substituted or unsubstituted heteroaryl. In thisclass of compounds, if

is a double bond, A¹, A², A⁴, A⁵, A⁶, A⁷, A⁸, and A¹⁰ are CH, A³ is—CCO₂H, R⁴, R⁷, and R⁸ are each hydrogen, R⁵ and R⁶ are methyl, and R³is cyano, then A⁹ is not —CBr.

Also provided herein are compositions including one or more compounds asdescribed above and a pharmaceutically acceptable carrier.

Further provided herein are methods of treating or preventing amicrobial infection in a subject. In some embodiments, the methodscomprise administering to the subject an effective amount of an RNaseinhibitor of the following structure:

or pharmaceutically acceptable salts or prodrugs thereof. In thesecompounds,

is a single or double bond; A¹, A², A³, A⁴, and A⁵, are eachindependently selected from N or CR¹; A⁶, A⁷, A⁸, A⁹, and A¹⁰ are eachindependently selected from N or CR²; each R¹, each R², R³, R⁵, R⁶, R⁷,and R⁸ are independently selected from hydrogen, halogen, hydroxyl,cyano, nitro, substituted or unsubstituted amino, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedheteroalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted heteroalkynyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedalkoxyl, substituted or unsubstituted aryloxyl, or substituted orunsubstituted carboxyl; and R⁴ is hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted heteroalkenyl,substituted or unsubstituted alkynyl, or substituted or unsubstitutedheteroalkyl. Optionally, R⁶ and R⁷ can combine to form substituted orunsubstituted aryl or substituted or unsubstituted heteroaryl. In someexamples, the RNase inhibitor is

In some embodiments, the microbial infection is a bacterial infection.The bacterial infection can be, for example, a Gram positive bacterialinfection. Optionally, the bacterial infection is a Staphylococcusinfection such as, for example, a Staphylococcus aureus infection. TheStaphylococcus aureus infection can be a drug-resistant Staphylococcusaureus infection or a biofilm-associated Staphylococcus aureusinfection. In some examples, the RNase inhibitor is a RnpA inhibitor.Optionally, the methods can further comprise administering a secondcompound to the subject, wherein the second compound is an antibacterialcompound.

Also provided herein are methods of inhibiting a bacterial ribonucleasecomprising contacting the bacterial ribonuclease with an effectiveamount of an RNase inhibitor. In some embodiments, the RNase inhibitoris a compound of the following structure:

or pharmaceutically acceptable salts or prodrugs thereof. In thesecompounds,

is a single or double bond; A¹, A², A³, A⁴, and A⁵, are eachindependently selected from N or CR¹; A⁶, A⁷, A⁸, A⁹, and A¹⁰ are eachindependently selected from N or CR²; each R¹, each R², R³, R⁵, R⁶, R⁷,and R⁸ are independently selected from hydrogen, halogen, hydroxyl,cyano, nitro, substituted or unsubstituted amino, substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl,substituted or unsubstituted alkenyl, substituted or unsubstitutedheteroalkenyl, substituted or unsubstituted alkynyl, substituted orunsubstituted heteroalkynyl, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedalkoxyl, substituted or unsubstituted aryloxyl, or substituted orunsubstituted carboxyl; and R⁴ is hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted heteroalkenyl,substituted or unsubstituted alkynyl, or substituted or unsubstitutedheteroalkyl. Optionally, R⁶ and R⁷ combine to form substituted orunsubstituted aryl or substituted or unsubstituted heteroaryl.

Optionally, the bacterial ribonuclease is the protein component ofStaphylococcus aureus RNase P (e.g., RnpA). The contacting can occur,for example, in vivo or in vitro.

Further provided herein are methods of identifying a compound fortreating or preventing a microbial infection. The method includes thesteps of combining RNA, RnpA, and a fluorescent dye to form a mixture;contacting the mixture with the compound; and monitoring RnpA-mediatedtotal bacterial RNA degradation in the cell using fluorescence, whereindecreased fluorescence, as compared to a control, indicates RNAdegradation. In this method, a compound that decreases the RnpA-mediatedtotal bacterial RNA degradation, as compared to a control, is identifiedas the compound for treating or preventing the microbial infection.

Additional advantages will be set forth in part in the description thatfollows, and in part will be obvious from the description, or may belearned by practice of the aspects described below. The advantagesdescribed below will be realized and attained by means of the elementsand combinations particularly pointed out in the appended claims. It isto be understood that both the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the percent of detectible mRNA species(Y-axis) with a half life of ≦2.5, 5, 15, 30, or >30 min duringexponential- and/or stationary-phase growth (X-axis).

FIG. 2 demonstrates that S. aureus RnpA catalyzes rRNA and mRNAdigestion. Panel A is an SDS-PAGE of purified recombinant S. aureusRnpA; shown are molecular markers (Lanes M), 2.5 μg and 25 μg elutionproducts (Lanes 1 and 2, respectively). Panel B depicts the gel-mobilityof 1 μg of total S. aureus RNA following 60 min incubation in theabsence (−) or presence (+) of 50 pmol of each putative ribonuclease(indicated) in 1× reaction buffer (2 mM NaCl, 2 mM MgCl₂, 50 mMTris-HCl, pH 6.0). Panel C displays the mobility of 0.5 pmol in vitrotranscribed spa mRNA following 60 min incubation in the absence (0 pmol)or presence of the indicated amount of RnpA protein in 1× reactionbuffer. Molecular weight markers (M) are shown. Panel D showsreverse-transcription mediated PCR products of 2 μg of in vitrotranscribed spa mRNA in the absence (−) or presence (+) of 50 pmol RnpAor RNase J1 and in the absence (serum alone) or presence of 1, 2.5, 5,10, or 20 μg RnpA polyclonal antibody. Panel E shows plottedmeasurements for all mRNA species measured on a GeneChip at 0 (X-axis)and 10 min (Y-axis) post-transcriptional arrest. Grey dashed lineindicates the lower limit of sensitivity for each sample.

FIG. 3, Panel A shows representative screening effort results. Panel Bshows an agarose gel-based assay depicting the gel mobility of molecularweight marker, spa mRNA in the absence (−) or presence (+) of 20 pmolRnpA and RnpA-mediated spa mRNA degradation in the presence ofincreasing concentrations of RNPA1000. Panel C shows the structure ofRnpA-inhibitory molecule RNPA1000.

FIG. 4, Panel A shows the MTT-cytotoxicity assay results of HepG2 cellsexposed to compound solvent (DMSO; negative control), Mitomycin C(positive control), and indicated amount of RNPA1000. Panel B shows theaverage daily (X-axis) percent surviving animals (Y-axis) following notreatment (closed diamonds; negative control), vancomycin treatment(closed squares; 1 mg/kg; positive control), or RNPA1000 treatment; 64mg/kg (open circles); 128 mg/kg (open squares), 256 mg/kg (opentriangles). Panel C shows the number of catheter-associated S. aureusfollowing 1, 2, or 3 days of no antimicrobial treatment (untreated; UT)or exposure to 5, 10, or 20 times the MIC for RNPA1000. Boxes define theinterval between the 25^(th) and 75^(th) percentile. Bars extendingupward indicate the boundary defined by the value 1.5× higher than the75^(th) percentile while those extending downward indicate the boundarydefined by a value 1.5× lower than the 25^(th) percentile. Filledcircles indicate individual values outside these two extremes.

FIG. 5 depicts a plot of all GeneChip detected transcript levels at 0and 5 min post-transcriptional arrest. Grey dashed line indicates thelower limit of sensitivity for each sample. Panel A shows a comparisonof the mRNA levels of DMSO treated cells (Left Panel) and a comparisonof the mRNA levels of cells challenged with sub-inhibitory concentrationof RnpA-inhibitor (Right Panel). Panel B shows a microtiter plate assayillustrating the in vitro antimicrobial effects of indicatedconcentration of RNPA1000 (across top) against S. aureus RN4220pRNPA-A.S. (RnpA depleted cells; top panel), RN4220 pRNPA (RnpAoverexpressor cells; center panel) and RN4220 pCN51 (vector; bottompanel) when grown in the presence of 2.5 μM CdCl₂. All strains wereassessed twice; arrows indicate MIC values.

FIG. 6, Panel A depicts plots of the growth characteristics (opticaldensity; Y-axis), for S. aureus strain RN4220 containing vector (pCN51;diamonds), rnpA sense RNA (pRNPA-S; triangles) and rnpA antisense RNA(pRNPA-A.S.; squares) when grown in the presence of 10 μM CdCl₂. Panel Bshows the Western blotting results for S. aureus strain RN4220 pCN51(vector), RN4220 pRNPA (overexpressor), and RN4220 pRNPA-A.S. (RnpAdepleted) cells grown in the presence of 2.5 μM CdCl₂.

FIG. 7 shows the S. aureus time-kill assay results. Panel A depicts themid-exponential phase for S. aureus strain UAMS-1 cells that weretreated with 0.25, 0.5, 1, 2, or 4 times the MIC for RNPA1000. Plottedare the average cfu/ml at 0, 2, 4, 8, and 24 hr post-RNPA1000 additionfor each drug concentration tested (n=3); standard deviation shown.Panel B shows the average cfu/ml at 2, 4, 8, and 24 hr post-oxacillintreatment (0.25, 0.5, 2, or 4 times the MIC; n=3) of mid-exponentialphase cells. Panel C shows the mid-exponential phase cells were treatedwith 0.5 times the MIC for RNPA1000, oxacillin, or both (RNPA1000 andoxacillin). Shown are the average cfu/ml of mid exponential phase cellsfollowing 2, 4, 8, and 24 hr post treatment (n=3); standard deviationshown.

FIG. 8 is a table showing the alignment of amino acid sequences of RnpAusing GramAlign with default parameters. Conserved amino acids areboxed. The sequence for S. aureus is SEQ ID NO. 1; for S. epidermidis isSEQ ID NO. 2; for S. pneumonia is SEQ ID NO. 3; for S. pyogenes is SEQID NO. 4; for E. faecalis is SEQ ID NO. 5; for E. coli is SEQ ID NO. 6;and for A. baumannii is SEQ ID NO. 7.

DETAILED DESCRIPTION

Provided herein are small molecule inhibitors of bacterial RnpAassociated ribonuclease (RNase) activity, methods of their preparation,and methods of their use in treating and preventing microbialinfections. The small molecule inhibitors exploit a novel mechanism oftreating microbial infections, such as Staphylococcus aureus, whichinvolves the essential S. aureus protein, RnpA, catalyzing rRNA and mRNAdigestion. This mechanism has not previously been known or developed.Exploiting this activity, high through-put and secondary screeningassays were employed to identify small molecule inhibitors ofRnpA-mediated RNA degradation. These agents limited cellular mRNAdegradation and exhibited antimicrobial activity against severalmicrobes, including predominant methicillin-resistant S. aureus (MRSA)lineages circulating throughout the U.S., vancomycin intermediatesusceptible S. aureus (VISA), vancomycin resistant S. aureus (VRSA) andother Gram-positive bacterial pathogens with high RnpA amino acidconservation (see McDougal et al., J Clin Microbiol, 41: 5113-5120(2003)). As provided herein, the RnpA-inhibitors limit disease in asystemic mouse infection model and have antimicrobial activity againstbiofilm-associated S. aureus. Taken together, these findings indicatethat RnpA plays a role in S. aureus RNA degradation, demonstrate thathigh through-put screening can be used to identify mRNA turnoverinhibitors, and provide proof of principle for RNA catabolism-basedantimicrobial therapy.

The materials, compounds, compositions, articles, and methods describedherein may be understood more readily by reference to the followingdetailed description of specific aspects of the disclosed subject matterand the Examples included therein.

Before the present materials, compounds, compositions, kits, and methodsare disclosed and described, it is to be understood that the aspectsdescribed below are not limited to specific synthetic methods orspecific reagents, as such may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular aspects only and is not intended to be limiting.

Also, throughout this specification, various publications arereferenced. The disclosures of these publications in their entiretiesare hereby incorporated by reference into this application in order tomore fully describe the state of the art to which the disclosed matterpertains. The references disclosed are also individually andspecifically incorporated by reference herein for the material containedin them that is discussed in the sentence in which the reference isrelied upon.

GENERAL DEFINITIONS

In this specification and in the claims that follow, reference will bemade to a number of terms, which shall be defined to have the followingmeanings:

Throughout the description and claims of this specification the word“comprise” and other forms of the word, such as “comprising” and“comprises,” means including but not limited to, and is not intended toexclude, for example, other additives, components, integers, or steps.

As used in the description and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a composition”includes mixtures of two or more such compositions, reference to “thecompound” includes mixtures of two or more such compounds, and the like.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another aspect includes from the one particular value and/orto the other particular value. Similarly, when values are expressed asapproximations, by use of the antecedent “about,” it will be understoodthat the particular value forms another aspect. It will be furtherunderstood that the endpoints of each of the ranges are significant bothin relation to the other endpoint, and independently of the otherendpoint. It is also understood that there are a number of valuesdisclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed, then“less than or equal to” the value, “greater than or equal to the value,”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed, then “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that throughoutthe application data are provided in a number of different formats andthat this data represent endpoints and starting points and ranges forany combination of the data points. For example, if a particular datapoint “10” and a particular data point “15” are disclosed, it isunderstood that greater than, greater than or equal to, less than, lessthan or equal to, and equal to 10 and 15 are considered disclosed aswell as between 10 and 15. It is also understood that each unit betweentwo particular units are also disclosed. For example, if 10 and 15 aredisclosed, then 11, 12, 13, and 14 are also disclosed.

As used herein, by a “subject” is meant an individual. Thus, the“subject” can include domesticated animals (e.g., cats, dogs, etc.),livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratoryanimals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds.“Subject” can also include a mammal, such as a primate or a human.

By “reduce” or other forms of the word, such as “reducing” or“reduction,” is meant lowering of an event or characteristic (e.g.,bacterial infection). It is understood that this is typically inrelation to some standard or expected value, in other words it isrelative, but that it is not always necessary for the standard orrelative value to be referred to. For example, “reduces bacterialinfection” means reducing the spread of a bacterial infection relativeto a standard or a control.

By “prevent” or other forms of the word, such as “preventing” or“prevention,” is meant to stop a particular event or characteristic, tostabilize or delay the development or progression of a particular eventor characteristic, or to minimize the chances that a particular event orcharacteristic will occur. Prevent does not require comparison to acontrol as it is typically more absolute than, for example, reduce. Asused herein, something could be reduced but not prevented, but somethingthat is reduced could also be prevented. Likewise, something could beprevented but not reduced, but something that is prevented could also bereduced. It is understood that where reduce or prevent are used, unlessspecifically indicated otherwise, the use of the other word is alsoexpressly disclosed.

By “treat” or other forms of the word, such as “treated” or “treatment,”is meant to administer a composition or to perform a method in order toreduce, prevent, inhibit, or eliminate a particular characteristic orevent (e.g., bacterial infection). The term “control” is usedsynonymously with the term “treat.”

By “antimicrobial” is meant the ability to treat or control (e.g.,reduce, prevent, inhibit, or eliminate) the growth of a microbe at anyconcentration. Similarly, the term “antibacterial” refers to the abilityto treat or control cellular bacteria growth at any concentration.

It is understood that throughout this specification the identifiers“first” and “second” are used solely to aid in distinguishing thevarious components and steps of the disclosed subject matter. Theidentifiers “first” and “second” are not intended to imply anyparticular order, amount, preference, or importance to the components orsteps modified by these terms.

CHEMICAL DEFINITIONS

As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, and aromatic and nonaromaticsubstituents of organic compounds. Illustrative substituents include,for example, those described below. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this disclosure, the heteroatoms, such as nitrogen, canhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valencies of theheteroatoms. This disclosure is not intended to be limited in any mannerby the permissible substituents of organic compounds. Also, the terms“substitution” or “substituted with” include the implicit proviso thatsuch substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., a compound that does not spontaneouslyundergo transformation such as by rearrangement, cyclization,elimination, etc.

“Z¹,” “Z²,” “Z³,” and “Z⁴” are used herein as generic symbols torepresent various specific substituents. These symbols can be anysubstituent, not limited to those disclosed herein, and when they aredefined to be certain substituents in one instance, they can, in anotherinstance, be defined as some other substituents.

The term “aliphatic” as used herein refers to a non-aromatic hydrocarbongroup and includes branched and unbranched, alkyl, alkenyl, or alkynylgroups.

The term “alkyl” as used herein is a branched or unbranched saturatedhydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl,n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl,octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl,tetracosyl, and the like. The alkyl group can also be substituted orunsubstituted. The alkyl group can be substituted with one or moregroups including, but not limited to, alkyl, halogenated alkyl, alkoxy,alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid,ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo,sulfonyl, sulfone, sulfoxide, or thiol, as described below.

Throughout the specification “alkyl” is generally used to refer to bothunsubstituted alkyl groups and substituted alkyl groups; however,substituted alkyl groups are also specifically referred to herein byidentifying the specific substituent(s) on the alkyl group. For example,the term “halogenated alkyl” specifically refers to an alkyl group thatis substituted with one or more halide, e.g., fluorine, chlorine,bromine, or iodine. The term “alkoxyalkyl” specifically refers to analkyl group that is substituted with one or more alkoxy groups, asdescribed below. The term “alkylamino” specifically refers to an alkylgroup that is substituted with one or more amino groups, as describedbelow, and the like. When “alkyl” is used in one instance and a specificterm such as “alkylalcohol” is used in another, it is not meant to implythat the term “alkyl” does not also refer to specific terms such as“alkylalcohol” and the like.

This practice is also used for other groups described herein. That is,while a term such as “cycloalkyl” refers to both unsubstituted andsubstituted cycloalkyl moieties, the substituted moieties can, inaddition, be specifically identified herein; for example, a particularsubstituted cycloalkyl can be referred to as, e.g., an“alkylcycloalkyl.” Similarly, a substituted alkoxy can be specificallyreferred to as, e.g., a “halogenated alkoxy,” a particular substitutedalkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, thepractice of using a general term, such as “cycloalkyl,” and a specificterm, such as “alkylcycloalkyl,” is not meant to imply that the generalterm does not also include the specific term.

The term “alkoxy” as used herein is an alkyl group bound through asingle, terminal ether linkage; that is, an “alkoxy” group can bedefined as —OZ¹ where Z¹ is alkyl as defined above.

The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon double bond. Asymmetric structures such as (Z¹Z²)C═C(Z³Z⁴)are intended to include both the E and Z isomers. This can be presumedin structural formulae herein wherein an asymmetric alkene is present,or it can be explicitly indicated by the bond symbol C═C. The alkenylgroup can be substituted with one or more groups including, but notlimited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl,heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide,or thiol, as described below.

The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24carbon atoms with a structural formula containing at least onecarbon-carbon triple bond. The alkynyl group can be substituted with oneor more groups including, but not limited to, alkyl, halogenated alkyl,alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylicacid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo,sulfonyl, sulfone, sulfoxide, or thiol, as described below.

The term “aryl” as used herein is a group that contains any carbon-basedaromatic group including, but not limited to, benzene, naphthalene,phenyl, biphenyl, phenoxybenzene, and the like. The term “heteroaryl” isdefined as a group that contains an aromatic group that has at least oneheteroatom incorporated within the ring of the aromatic group. Examplesof heteroatoms include, but are not limited to, nitrogen, oxygen,sulfur, and phosphorus. The term “non-heteroaryl,” which is included inthe term “aryl,” defines a group that contains an aromatic group thatdoes not contain a heteroatom. The aryl or heteroaryl group can besubstituted or unsubstituted. The aryl or heteroaryl group can besubstituted with one or more groups including, but not limited to,alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl,aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone,nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol asdescribed herein. The term “biaryl” is a specific type of aryl group andis included in the definition of aryl. Biaryl refers to two aryl groupsthat are bound together via a fused ring structure, as in naphthalene,or are attached via one or more carbon-carbon bonds, as in biphenyl.

The term “cycloalkyl” as used herein is a non-aromatic carbon-based ringcomposed of at least three carbon atoms. Examples of cycloalkyl groupsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, etc. The term “heterocycloalkyl” is a cycloalkyl group asdefined above where at least one of the carbon atoms of the ring issubstituted with a heteroatom such as, but not limited to, nitrogen,oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkylgroup can be substituted or unsubstituted. The cycloalkyl group andheterocycloalkyl group can be substituted with one or more groupsincluding, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl,heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide,or thiol as described herein.

The term “cycloalkenyl” as used herein is a non-aromatic carbon-basedring composed of at least three carbon atoms and containing at least onedouble bound, i.e., C═C. Examples of cycloalkenyl groups include, butare not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl,cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like. The term“heterocycloalkenyl” is a type of cycloalkenyl group as defined above,and is included within the meaning of the term “cycloalkenyl,” where atleast one of the carbon atoms of the ring is substituted with aheteroatom such as, but not limited to, nitrogen, oxygen, sulfur, orphosphorus. The cycloalkenyl group and heterocycloalkenyl group can besubstituted or unsubstituted. The cycloalkenyl group andheterocycloalkenyl group can be substituted with one or more groupsincluding, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl,heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide,hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide,or thiol as described herein.

The term “cyclic group” is used herein to refer to either aryl groups,non-aryl groups (i.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl groups), or both. Cyclic groups have one or more ringsystems that can be substituted or unsubstituted. A cyclic group cancontain one or more aryl groups, one or more non-aryl groups, or one ormore aryl groups and one or more non-aryl groups.

The term “aldehyde” as used herein is represented by the formula —C(O)H.Throughout this specification “C(O)” or “CO” is a short hand notationfor C═O.

The terms “amine” or “amino” as used herein are represented by theformula —NZ¹Z², where Z¹ and Z² can each be substitution group asdescribed herein, such as hydrogen, an alkyl, halogenated alkyl,alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl,heterocycloalkyl, or heterocycloalkenyl group described above.

The term “carboxylic acid” as used herein is represented by the formula—C(O)OH. A “carboxylate” or “carboxyl” group as used herein isrepresented by the formula —C(O)O⁻.

The term “ester” as used herein is represented by the formula —OC(O)Z¹or —C(O)OZ¹, where Z¹ can be an alkyl, halogenated alkyl, alkenyl,alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl,or heterocycloalkenyl group described above.

The term “ether” as used herein is represented by the formula A¹OA²,where A¹ and A² can be, independently, an alkyl, halogenated alkyl,alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl,heterocycloalkyl, or heterocycloalkenyl group described above.

The term “ketone” as used herein is represented by the formula Z¹C(O)Z²,where Z¹ and Z² can be, independently, an alkyl, halogenated alkyl,alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl,heterocycloalkyl, or heterocycloalkenyl group described above.

The term “halide” or “halogen” as used herein refers to the fluorine,chlorine, bromine, and iodine.

The term “hydroxyl” as used herein is represented by the formula —OH.

The term “nitro” as used herein is represented by the formula —NO₂.

The term “silyl” as used herein is represented by the formula —SiZ¹Z²Z³,where Z¹, Z², and Z³ can be, independently, hydrogen, alkyl, halogenatedalkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl,cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group describedabove.

The term “sulfonyl” is used herein to refer to the sulfo-oxo grouprepresented by the formula —S(O)₂Z¹, where Z¹ can be hydrogen, an alkyl,halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl,cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group describedabove.

The term “sulfonylamino” or “sulfonamide” as used herein is representedby the formula —S(O)₂NH—.

The term “thiol” as used herein is represented by the formula —SH.

The term “thio” as used herein is represented by the formula —S—.

“R¹,” “R²,” “R³,” “R^(n),” etc., where n is some integer, as used hereincan, independently, possess one or more of the groups listed above. Forexample, if R¹ is a straight chain alkyl group, one of the hydrogenatoms of the alkyl group can optionally be substituted with a hydroxylgroup, an alkoxy group, an amine group, an alkyl group, a halide, andthe like. Depending upon the groups that are selected, a first group canbe incorporated within second group or, alternatively, the first groupcan be pendant (i.e., attached) to the second group. For example, withthe phrase “an alkyl group comprising an amino group,” the amino groupcan be incorporated within the backbone of the alkyl group.Alternatively, the amino group can be attached to the backbone of thealkyl group. The nature of the group(s) that is (are) selected willdetermine if the first group is embedded or attached to the secondgroup.

Unless stated to the contrary, a formula with chemical bonds shown onlyas solid lines and not as wedges or dashed lines contemplates eachpossible isomer, e.g., each enantiomer, diastereomer, and meso compound,and a mixture of isomers, such as a racemic or scalemic mixture.

Reference will now be made in detail to specific aspects of thedisclosed materials, compounds, compositions, articles, and methods,examples of which are illustrated in the accompanying Examples.

Compounds

The small molecule inhibitors of bacterial ribonuclease (RNase)described herein include compounds represented by Formula I:

and pharmaceutically acceptable salts and prodrugs thereof

In Formula I,

is a single or double bond.

Also in Formula I, A¹, A², A³, A⁴, and A⁵ are each independentlyselected from N or CR¹. Each R¹ can be independently selected fromhydrogen, halogen, hydroxyl, cyano, nitro, substituted or unsubstitutedamino, substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted alkenyl, substituted orunsubstituted heteroalkenyl, substituted or unsubstituted alkynyl,substituted or unsubstituted heteroalkynyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted alkoxyl, substituted or unsubstituted aryloxyl, orsubstituted or unsubstituted carboxyl. Optionally, one or more of A¹,A², A³, A⁴, and A⁵ is CH. In some embodiments, A³ is —CCO₂H.

Additionally in Formula I, A⁶, A⁷, A⁸, A⁹, and A¹⁰ are eachindependently selected from N or CR². Each R² can be independentlyselected from hydrogen, halogen, hydroxyl, cyano, nitro, substituted orunsubstituted amino, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted alkenyl,substituted or unsubstituted heteroalkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted heteroalkynyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted alkoxyl, substituted or unsubstituted aryloxyl, orsubstituted or unsubstituted carboxyl. Optionally, one or more of A⁶,A⁷, A⁸, A⁹, and A¹⁰ is CH. In some embodiments, A⁹ is CBr. In someembodiments, A⁸ is CBr.

Also in Formula I, R³, R⁵, R⁶, R⁷, and R⁸ are independently selectedfrom hydrogen, halogen, hydroxyl, cyano, nitro, substituted orunsubstituted amino, substituted or unsubstituted alkyl, substituted orunsubstituted heteroalkyl, substituted or unsubstituted alkenyl,substituted or unsubstituted heteroalkenyl, substituted or unsubstitutedalkynyl, substituted or unsubstituted heteroalkynyl, substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl, substitutedor unsubstituted alkoxyl, substituted or unsubstituted aryloxyl, orsubstituted or unsubstituted carboxyl. Optionally, R³ is cyano. In someembodiments, R⁵ and R⁶ are methyl.

Further in Formula I, R⁴ is hydrogen, substituted or unsubstitutedalkyl, substituted or unsubstituted heteroalkyl, substituted orunsubstituted alkenyl, substituted or unsubstituted heteroalkenyl,substituted or unsubstituted alkynyl, or substituted or unsubstitutedheteroalkyl.

In Formula I, adjacent R groups, e.g., R⁶ and R⁷, can be combined toform a substituted or unsubstituted aryl, substituted or unsubstitutedheteroaryl, substituted or unsubstituted cycloalkyl, substituted orunsubstituted cycloalkenyl, substituted or unsubstituted cycloalkynyl,substituted or unsubstituted heterocycloalkyl, substituted orunsubstituted heterocycloalkenyl, or substituted or unsubstitutedheterocycloalkynyl. For example, R⁶ can be a substituted orunsubstituted ethylene group and R⁷ can be a substituted orunsubstituted propylene group that combine to form a substituted orunsubstituted phenyl. In these examples, R⁶ and R⁷ combine to formStructure I-A, i.e., the indole embodiments:

Optionally, the phenyl ring of the indole in Structure I-A can besubstituted with R⁹. In Structure I-A, R⁹ is selected from hydrogen,halogen, hydroxyl, cyano, nitro, substituted or unsubstituted amino,substituted or unsubstituted alkyl, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted alkenyl, substituted orunsubstituted heteroalkenyl, substituted or unsubstituted alkynyl,substituted or unsubstituted heteroalkynyl, substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted alkoxyl, substituted or unsubstituted aryloxyl, orsubstituted or unsubstituted carboxyl.

In some examples of Formula I, each of A¹, A², A³, A⁴, and A⁵ are CH toform Structure I-B. In other examples of Formula I, each of A⁶, A⁷, A⁸,A⁹, and A¹⁰ are CH to form Structure I-C. In still other examples ofFormula I, each of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ are CH toform Structure I-D.

Optionally, the compound according to Formula I includes an enone and isa compound according to Structure I-E. In some embodiments, the enone isreduced to form a compound according to Structure I-F.

In some embodiments, A³ in Formula I is —CCO₂H as shown in StructureI-G:

In some examples of Formula I, if

is a double bond, A¹, A², A⁴, A⁵, A⁶, A⁷, A⁸, and A¹⁰ are CH, A³ is—CCO₂H, R⁴, R⁷, and R⁸ are each hydrogen, R⁵ and R⁶ are methyl, and R³is cyano, then A⁹ is not —CBr.

A particular example of Formula I is compound RNPA-1000:

Pharmaceutical Compositions

The compounds described herein or derivatives thereof can be provided ina pharmaceutical composition. Depending on the intended mode ofadministration, the pharmaceutical composition can be in the form ofsolid, semi-solid or liquid dosage forms, such as, for example, tablets,suppositories, pills, capsules, powders, liquids, or suspensions,preferably in unit dosage form suitable for single administration of aprecise dosage. The compositions will include a therapeuticallyeffective amount of the compound described herein or derivatives thereofin combination with a pharmaceutically acceptable carrier and, inaddition, can include other medicinal agents, pharmaceutical agents,carriers, or diluents. By pharmaceutically acceptable is meant amaterial that is not biologically or otherwise undesirable, which can beadministered to an individual along with the selected compound withoutcausing unacceptable biological effects or interacting in a deleteriousmanner with the other components of the pharmaceutical composition inwhich it is contained.

As used herein, the term carrier encompasses any excipient, diluent,filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, orother material well known in the art for use in pharmaceuticalformulations. The choice of a carrier for use in a composition willdepend upon the intended route of administration for the composition.The preparation of pharmaceutically acceptable carriers and formulationscontaining these materials is described in, e.g., Remington'sPharmaceutical Sciences, 21st Edition, ed. University of the Sciences inPhiladelphia, Lippincott, Williams & Wilkins, Philadelphia Pa., 2005.Examples of physiologically acceptable carriers include buffers such asphosphate buffers, citrate buffer, and buffers with other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™ (ICI, Inc.; Bridgewater, N.J.), polyethylene glycol(PEG), and PLURONICS™ (BASF; Florham Park, N.J.).

Compositions containing the compound described herein or derivativesthereof suitable for parenteral injection can comprise physiologicallyacceptable sterile aqueous or nonaqueous solutions, dispersions,suspensions or emulsions, and sterile powders for reconstitution intosterile injectable solutions or dispersions. Examples of suitableaqueous and nonaqueous carriers, diluents, solvents or vehicles includewater, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol,and the like), suitable mixtures thereof, vegetable oils (such as oliveoil) and injectable organic esters such as ethyl oleate. Proper fluiditycan be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersions and by the use of surfactants.

These compositions can also contain adjuvants such as preserving,wetting, emulsifying, and dispensing agents. Prevention of the action ofmicroorganisms can be promoted by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, sorbic acid, andthe like. Isotonic agents, for example, sugars, sodium chloride, and thelike can also be included. Prolonged absorption of the injectablepharmaceutical form can be brought about by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Solid dosage forms for oral administration of the compounds describedherein or derivatives thereof include capsules, tablets, pills, powders,and granules. In such solid dosage forms, the compounds described hereinor derivatives thereof is admixed with at least one inert customaryexcipient (or carrier) such as sodium citrate or dicalcium phosphate or(a) fillers or extenders, as for example, starches, lactose, sucrose,glucose, mannitol, and silicic acid, (b) binders, as for example,carboxymethylcellulose, alignates, gelatin, polyvinylpyrrolidone,sucrose, and acacia, (c) humectants, as for example, glycerol, (d)disintegrating agents, as for example, agar-agar, calcium carbonate,potato or tapioca starch, alginic acid, certain complex silicates, andsodium carbonate, (e) solution retarders, as for example, paraffin, (f)absorption accelerators, as for example, quaternary ammonium compounds,(g) wetting agents, as for example, cetyl alcohol, and glycerolmonostearate, (h) adsorbents, as for example, kaolin and bentonite, and(i) lubricants, as for example, talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, or mixturesthereof. In the case of capsules, tablets, and pills, the dosage formscan also comprise buffering agents.

Solid compositions of a similar type can also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethyleneglycols, andthe like.

Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others known in the art. They can contain opacifying agentsand can also be of such composition that they release the activecompound or compounds in a certain part of the intestinal tract in adelayed manner. Examples of embedding compositions that can be used arepolymeric substances and waxes. The active compounds can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-mentioned excipients.

Liquid dosage forms for oral administration of the compounds describedherein or derivatives thereof include pharmaceutically acceptableemulsions, solutions, suspensions, syrups, and elixirs. In addition tothe active compounds, the liquid dosage forms can contain inert diluentscommonly used in the art, such as water or other solvents, solubilizingagents, and emulsifiers, as for example, ethyl alcohol, isopropylalcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzylbenzoate, propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils,in particular, cottonseed oil, groundnut oil, corn germ oil, olive oil,castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol,polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures ofthese substances, and the like.

Besides such inert diluents, the composition can also include additionalagents, such as wetting, emulsifying, suspending, sweetening, flavoring,or perfuming agents.

Suspensions, in addition to the active compounds, can contain additionalagents, as for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, or mixtures of thesesubstances, and the like.

Compositions of the compounds described herein or derivatives thereoffor rectal administrations are optionally suppositories, which can beprepared by mixing the compounds with suitable non-irritating excipientsor carriers such as cocoa butter, polyethyleneglycol or a suppositorywax, which are solid at ordinary temperatures but liquid at bodytemperature and therefore, melt in the rectum or vaginal cavity andrelease the active component.

Dosage forms for topical administration of the compounds describedherein or derivatives thereof include ointments, powders, sprays, andinhalants. The compounds described herein or derivatives thereof areadmixed under sterile conditions with a physiologically acceptablecarrier and any preservatives, buffers, or propellants as can berequired. Ophthalmic formulations, ointments, powders, and solutions arealso contemplated as being within the scope of the compositions.

The compositions can include one or more of the compounds describedherein and a pharmaceutically acceptable carrier. As used herein, theterm pharmaceutically acceptable salt refers to those salts of thecompound described herein or derivatives thereof that are, within thescope of sound medical judgment, suitable for use in contact with thetissues of subjects without undue toxicity, irritation, allergicresponse, and the like, commensurate with a reasonable benefit/riskratio, and effective for their intended use, as well as the zwitterionicforms, where possible, of the compounds described herein. The term saltsrefers to the relatively non-toxic, inorganic and organic acid additionsalts of the compounds described herein. These salts can be prepared insitu during the isolation and purification of the compounds or byseparately reacting the purified compound in its free base form with asuitable organic or inorganic acid and isolating the salt thus formed.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate,stearate, laurate, borate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate,glucoheptonate, lactobionate, methane sulphonate, and laurylsulphonatesalts, and the like. These can include cations based on the alkali andalkaline earth metals, such as sodium, lithium, potassium, calcium,magnesium, and the like, as well as non-toxic ammonium, quaternaryammonium, and amine cations including, but not limited to ammonium,tetramethylammonium, tetraethylammonium, methylamine, dimethylamine,trimethylamine, triethylamine, ethylamine, and the like. (See S. M.Barge et al., J. Pharm. Sci. (1977) 66, 1, which is incorporated hereinby reference in its entirety, at least, for compositions taught herein.)

Administration of the compounds and compositions described herein orpharmaceutically acceptable salts thereof to a subject can be carriedout using therapeutically effective amounts of the compounds andcompositions described herein or pharmaceutically acceptable saltsthereof as described herein for periods of time effective to treat adisorder.

The effective amount of the compounds and compositions described hereinor pharmaceutically acceptable salts thereof as described herein can bedetermined by one of ordinary skill in the art and includes exemplarydosage amounts for a mammal of from about 0.5 to about 200 mg/kg of bodyweight of active compound per day, which can be administered in a singledose or in the form of individual divided doses, such as from 1 to 4times per day. Alternatively, the dosage amount can be from about 0.5 toabout 150 mg/kg of body weight of active compound per day, about 0.5 to100 mg/kg of body weight of active compound per day, about 0.5 to about75 mg/kg of body weight of active compound per day, about 0.5 to about50 mg/kg of body weight of active compound per day, about 0.5 to about25 mg/kg of body weight of active compound per day, about 1 to about 20mg/kg of body weight of active compound per day, about 1 to about 10mg/kg of body weight of active compound per day, about 20 mg/kg of bodyweight of active compound per day, about 10 mg/kg of body weight ofactive compound per day, or about 5 mg/kg of body weight of activecompound per day. The expression effective amount, when used to describean amount of compound in a method, refers to the amount of a compoundthat achieves the desired pharmacological effect or other effect, forexample an amount that results in bacterial enzyme inhibition.

Those of skill in the art will understand that the specific dose leveland frequency of dosage for any particular subject can be varied andwill depend upon a variety of factors, including the activity of thespecific compound employed, the metabolic stability and length of actionof that compound, the species, age, body weight, general health, sex anddiet of the subject, the mode and time of administration, rate ofexcretion, drug combination, and severity of the particular condition.

Methods of Making the Compounds

The compounds described herein can be prepared in a variety of waysknown to one skilled in the art of organic synthesis or variationsthereon as appreciated by those skilled in the art. The compoundsdescribed herein can be prepared from readily available startingmaterials. Optimum reaction conditions can vary with the particularreactants or solvents used, but such conditions can be determined by oneskilled in the art.

Variations on Formula I include the addition, subtraction, or movementof the various constituents as described for each compound. Similarly,when one or more chiral centers are present in a molecule, the chiralityof the molecule can be changed. Additionally, compound synthesis caninvolve the protection and deprotection of various chemical groups. Theuse of protection and deprotection, and the selection of appropriateprotecting groups can be determined by one skilled in the art. Thechemistry of protecting groups can be found, for example, in Wuts andGreene, Protective Groups in Organic Synthesis, 4th Ed., Wiley & Sons,2006, which is incorporated herein by reference in its entirety.

The starting materials and reagents used in preparing the disclosedcompounds and compositions are either available from commercialsuppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), AcrosOrganics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.),Sigma (St. Louis, Mo.), Pfizer (New York, N.Y.), GlaxoSmithKline(Raleigh, N.C.), Merck (Whitehouse Station, N.J.), Johnson & Johnson(New Brunswick, N.J.), Aventis (Bridgewater, N.J.), AstraZeneca(Wilmington, Del.), Novartis (Basel, Switzerland), Wyeth (Madison,N.J.), Bristol-Myers-Squibb (New York, N.Y.), Roche (Basel,Switzerland), Lilly (Indianapolis, Ind.), Abbott (Abbott Park, Ill.),Schering Plough (Kenilworth, N.J.), or Boehringer Ingelheim (Ingelheim,Germany), or are prepared by methods known to those skilled in the artfollowing procedures set forth in references such as Fieser and Fieser'sReagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons,1991); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 andSupplementals (Elsevier Science Publishers, 1989); Organic Reactions,Volumes 1-40 (John Wiley and Sons, 1991); March's Advanced OrganicChemistry, (John Wiley and Sons, 4th Edition); and Larock'sComprehensive Organic Transformations (VCH Publishers Inc., 1989). Othermaterials, such as the pharmaceutical carriers disclosed herein can beobtained from commercial sources.

Reactions to produce the compounds described herein can be carried outin solvents, which can be selected by one of skill in the art of organicsynthesis. Solvents can be substantially nonreactive with the startingmaterials (reactants), the intermediates, or products under theconditions at which the reactions are carried out, i.e., temperature andpressure. Reactions can be carried out in one solvent or a mixture ofmore than one solvent. Product or intermediate formation can bemonitored according to any suitable method known in the art. Forexample, product formation can be monitored by spectroscopic means, suchas nuclear magnetic resonance spectroscopy (e.g., ¹H or ¹³C) infraredspectroscopy, spectrophotometry (e.g., UV-visible), or massspectrometry, or by chromatography such as high performance liquidchromatography (HPLC) or thin layer chromatography.

Analogs of RNAP 1000 with various diversity groups can be prepared usinga number of known synthetic schemes (Jendralla et al., J. Med. Chem.,33(1): 61-70 (1990)), including the reaction steps shown in Scheme 1.The condensation shown in Step 1 can be accelerated using microwavetechnology (He et al., J. Org. Chem., 7:1150-1157 (2011)). Incorporationof a leaving group such as bromo into the R¹ position shown in Scheme 1allows for further homologation via transition metal-catalyzed chemistrysuch as Suzuki and Buchwald condensations. Mixtures of stereoisomersmight be expected from the Knoevenagal reaction shown in Step 3, but theisomers can be separated via chromatography and the corresponding (Z)-and (E)-isomers can be separately converted to final targets, enhancingthe diversity set.

Additional diversity can be obtained by varying the reagents in thegeneral scheme. Substitution of propane-2,5-dione with homologous dioneswill allow for exploration of additional binding interactions adjacentto the pyrrole core (Scheme 2). The Vilsmeier-Haack formylation can givea mixture of regioisomers, which can be separated by chromatography(Manetti et al., Chem Med Chem, 1(9): 973-989 (2006)) and convertedseparately to final target compounds. Indole analogs can be preparedfrom known indole-3-carboxaldehydes using an analogous scheme (Khan etal., Journal of Heterocyclic Chemistry, 16(5): 997-999 (1979)).

The role of the nitrile can be investigated by substitutingmalononitrile with a protected malonic diester that is resistant totransamination, such as the t-butyl ester shown in Scheme 3.Deprotection provides the intermediate acid, which can be furtherelaborated to explore the SAR around that region of the molecule.Finally, reduction of the double bond of the adduct obtained from theKnoevenagal reactions (e.g., Scheme 3, step 3) provides analogs with andwithout enone moieties. Racemic mixtures can be tested by chiral HPLC,and subsequently separated.

Activity Assays

Provided herein are methods of identifying a compound for treating orpreventing a microbial infection. The methods can include preparing acompound or composition as described herein and assaying the inhibitoryactivity of the compound or composition against bacterial ribonucleases,such as RNase P. RNase P is an ubiquitous enzyme that catalyzesmaturation of the 5′ end of precursor tRNAs (see Frank et al., Annu RevBiochem, 67: 153-180 (1998); Kazantsev et al., Nat Rev Microbiol, 4:729-740 (2006); Walker et al., Crit Rev Biochem Mol Biol, 41: 77-102(2006)). The enzyme is unique by virtue of the fact that it is aribonucleoprotein complex, which includes a single ribozyme RNA moleculeand at least one protein component. Within bacteria both the ribozyme(rnpB) and protein (RnpA) components are required for cell viability;rnpB mediates tRNA processing in vitro, whereas no function has beenfirmly established for RnpA (see Gossringer et al., J Bacteriol, 188:6816-6823 (2006); Schedl et al., Proc Natl Acad Sci US A, 70: 2091-2095(1973); Waugh et al., J Bacteriol, 172: 6316-6322 (1990)). Domainsearches (see Letunic et al., Nucleic Acids Res, 34: D257-260 (2006);Schultz et al., Proc Natl Acad Sci USA, 95: 5857-5864 (1998)) revealedthat S. aureus RnpA residues 40-111 best conform to a ribonuclease-likemotif. Further, several RNA binding sites are embedded within thisregion (see Spitzfaden et al., J Mol Biol, 295: 105-115 (2000)). E. coliand B. subtilis RNase P have been found to digest certaindouble-stranded RNA templates, such as guide-RNAs and 4.5s RNA (seeLundblad et al., Proc Natl Acad Sci USA, 105: 2354-2357 (2008)).Cleavage of those templates strictly requires RnpA (see Liu et al.,Cell, 77: 1093-1100 (1994); Marvin et al., J Cell Biochem, 108:1244-1251 (2009)). As provided herein, RNase P mediated RNA digestionmay be dependent on rnpB, RnpA, or both. Thus, RnpA modulates S. aureusRNA degradation.

RNA degradation can be used to identify compounds suitable inhibitingbacterial ribonucleases, and thus, suitable for treating or preventing amicrobial infection. In some embodiments, a fluorescence based assay canbe used to identify the compounds. The method can include the steps ofcombining RNA, RnpA, and a fluorescent dye to form a mixture, contactingthe mixture with the compound, and monitoring RnpA-mediated totalbacterial RNA degradation in the cell using fluorescence. Decreasedfluorescence, as compared to a control, indicates RNA degradation. Asused herein, decreased fluorescence refers to a lowering offluorescence, as compared to a control, of at least about 1%. Forexample, decreased fluorescence can be a decrease in fluorescence of atleast about 5%, at least about 10%, at least about 15%, at least about20%, at least about 25%, at least about 30%, at least about 35%, atleast about 40%, at least about 45%, at least about 50%, at least about55%, at least about 60%, at least about 65%, at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or at least about 99%, as compared to acontrol. A compound that decreases the RnpA-mediated total bacterial RNAdegradation, as compared to a control, can be identified as the compoundfor treating or preventing the microbial infection. A suitablefluorescent dye for use in the methods described herein includesQuant-iT RiboGreen® (Invitrogen; Carlsbad, Calif.).

In some examples, compounds can be further assayed using the MuellerHinton (MH) broth antibacterial assay as specified by the Clinical andLaboratory Standards Institute MIC broth microdilution protocol (seeMethods for Dilution Antimicrobial Susceptibility Tests for BacteriaThat Grow Aerobically; Approved Standard, In The Clinical and LaboratoryStandards Institute (CLSI, formerly NCCLS), 7^(th) ed., January 2006, 26(2), M7-A7; see also Performance Standards for AntimicrobialSusceptibility Testing; Eighteenth Informational Supplement, In TheClinical and Laboratory Standards Institute (CLSI, formerly NCCLS),January 2008, 28 (1), M100-S18).

The activity of the compounds and compositions provided herein asinhibitors of bacterial RNase can be measured in standard assays, e.g.,HPLC assays. The compounds can be tested as inhibitors of bacterialRNase in a bacterial RNase enzyme assay. Compounds that are identifiedas bacterial RNase inhibitors are useful in treating or preventingmicrobial infections. The activities of the compounds and compositionsas determined using the assays can be reported in terms of IC₅₀. As usedherein, IC₅₀ refers to an amount, concentration, or dosage of aparticular test compound that achieves a 50% inhibition of a maximalresponse in an assay that measures such response.

In certain aspects, the disclosed compounds and compositions need notactually be synthesized, but instead can be used as targets for anymolecular modeling technique to predict and characterize interactionswith bacterial RNase. This is achieved through structural informationand computer modeling. Computer modeling technology allows visualizationof the three-dimensional atomic structure of a selected molecule and therational design of new compounds that will interact with the enzyme. Thethree-dimensional construct of the enzyme typically depends on data fromx-ray crystallographic analyses or NMR imaging of the selected molecule.This data is available for bacterial RNase. The molecular dynamicsrequire force field data (e.g., Merck Molecular Force Field). Thecomputer graphics systems enable prediction of how a new compound willlink to the enzyme and allow experimental manipulation of the structuresof the compound to perfect binding specificity. Prediction of what theinteractions will be when small changes are made in one or both requiresmolecular mechanics software and computationally intensive computers,usually coupled with user-friendly, menu-driven interfaces between themolecular design program and the user.

Examples of molecular modeling systems are the CHARMm and QUANTAprograms, Polygen Corporation, Waltham, Mass. CHARMm performs the energyminimization and molecular dynamics functions. QUANTA performs theconstruction, graphic modeling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other. Uponidentification of compounds that interact in a desired way withbacterial RNase in silico, actual compounds can be synthesized andassayed as disclosed herein.

Methods of Use

Provided herein are methods to treat, prevent, or limit microbialinfections in a subject. The methods include administering to a subjectan effective amount of one or more of the compounds or compositionsdescribed herein, or a pharmaceutically acceptable salt thereof. Thecompounds and compositions described herein or pharmaceuticallyacceptable salts thereof are useful for treating microbial infectionsand cancer in humans, e.g., pediatric and geriatric populations, and inanimals, e.g., veterinary applications. Microbial infections include,for example, bacterial and fungal infections. Bacterial infectionsinclude infections caused by bacilli, cocci, spirochaetes, and vibriobacteria. In some examples, the microbial infection is a bacterialinfection (e.g., a Gram positive bacterial infection). In some examples,the bacterial infection is Staphylococcus infection, such as aStaphylococcus aureus. The compounds and compositions described hereinare useful in treating a variety of Staphylococcus aureus infections,including drug-resistant Staphylococcus aureus infections andbiofilm-associated Staphylococcus aureus infections. In someembodiments, the Staphylococcus aureus infection ismethocillin-resistant S. aureus (S. aureus MRSA). In other embodiments,the Staphylococcus aureus infection is vancomycin-resistant S. aureus.Optionally, the Staphylococcus aureus infection is multi-drug resistant.In some examples, the compounds and compositions described herein can beused to treat Bacillus infections (e.g., Bacillus anthracis and Bacilluscereus), Streptococcus infections (e.g., Streptococcus pneumoniae andStreptococcus pyogenes), and Enterococcus infections (e.g., Enterococcusfaecalis and vancomycin-resistant Enterococcus).

The methods of treatment or prevention described herein can furtherinclude treatment with one or more additional agents (e.g., anantibacterial agent). The one or more additional agents and thecompounds and compositions or pharmaceutically acceptable salts thereofas described herein can be administered in any order, includingsimultaneous administration, as well as temporally spaced order of up toseveral days apart. The methods can also include more than a singleadministration of the one or more additional agents and/or the compoundsand compositions or pharmaceutically acceptable salts thereof asdescribed herein. The administration of the one or more additionalagents and the compounds and compositions or pharmaceutically acceptablesalts thereof as described herein can be by the same or differentroutes. When treating with one or more additional agents, the compoundsand compositions or pharmaceutically acceptable salts thereof asdescribed herein can be combined into a pharmaceutical composition thatincludes the one or more additional agents. For example, the compoundsor compositions or pharmaceutically acceptable salts thereof asdescribed herein can be combined into a pharmaceutical composition withan additional antibacterial agent, such as acedapsone; acetosulfonesodium; alamecin; alexidine; amdinocillin; amdinocillin pivoxil;amicycline; amifloxacin; amifloxacin mesylate; amikacin; amikacinsulfate; aminosalicylic acid; aminosalicylate sodium; amoxicillin;amphomycin; ampicillin; ampicillin sodium; apalcillin sodium; apramycin;aspartocin; astromicin sulfate; avilamycin; avoparcin; azithromycin;azlocillin; azlocillin sodium; bacampicillin hydrochloride; bacitracin;bacitracin methylene disalicylate; bacitracin zinc; bambermycins;benzoylpas calcium; berythromycin; betamicin sulfate; biapenem;biniramycin; biphenamine hydrochloride; bispyrithione magsulfex;butikacin; butirosin sulfate; capreomycin sulfate; carbadox;carbenicillin disodium; carbenicillin indanyl sodium; carbenicillinphenyl sodium; carbenicillin potassium; carumonam sodium; cefaclor;cefadroxil; cefamandole; cefamandole nafate; cefamandole sodium;cefaparole; cefatrizine; cefazaflur sodium; cefazolin; cefazolin sodium;cefbuperazone; cefdinir; cefepime; cefepime hydrochloride; cefetecol;cefixime; cefmenoxime hydrochloride; cefmetazole; cefmetazole sodium;cefonicid monosodium; cefonicid sodium; cefoperazone sodium; ceforanide;cefotaxime sodium; cefotetan; cefotetan disodium; cefotiamhydrochloride; cefoxitin; cefoxitin sodium; cefpimizole; cefpimizolesodium; cefpiramide; cefpiramide sodium; cefpirome sulfate; cefpodoximeproxetil; cefprozil; cefroxadine; cefsulodin sodium; ceftazidime;ceftibuten; ceftizoxime sodium; ceftriaxone sodium; cefuroxime;cefuroxime axetil; cefuroxime pivoxetil; cefuroxime sodium; cephacetrilesodium; cephalexin; cephalexin hydrochloride; cephaloglycin;cephaloridine; cephalothin sodium; cephapirin sodium; cephradine;cetocycline hydrochloride; cetophenicol; chloramphenicol;chloramphenicol palmitate; chloramphenicol pantothenate complex;chloramphenicol sodium succinate; chlorhexidine phosphanilate;chloroxylenol; chlortetracycline bisulfate; chlortetracyclinehydrochloride; cinoxacin; ciprofloxacin; ciprofloxacin hydrochloride;cirolemycin; clarithromycin; clinafloxacin hydrochloride; clindamycin;clindamycin hydrochloride; clindamycin palmitate hydrochloride;clindamycin phosphate; clofazimine; cloxacillin benzathine; cloxacillinsodium; cloxyquin; colistimethate sodium; colistin sulfate; coumermycin;coumermycin sodium; cyclacillin; cycloserine; dalfopristin; dapsone;daptomycin; demeclocycline; demeclocycline hydrochloride; demecycline;denofungin; diaveridine; dicloxacillin; dicloxacillin sodium;dihydrostreptomycin sulfate; dipyrithione; dirithromycin; doxycycline;doxycycline calcium; doxycycline fosfatex; doxycycline hyclate; droxacinsodium; enoxacin; epicillin; epitetracycline hydrochloride;erythromycin; erythromycin acistrate; erythromycin estolate;erythromycin ethylsuccinate; erythromycin gluceptate; erythromycinlactobionate; erythromycin propionate; erythromycin stearate; ethambutolhydrochloride; ethionamide; fleroxacin; floxacillin; fludalanine;flumequine; fosfomycin; fosfomycin tromethamine; fumoxicillin;furazolium chloride; furazolium tartrate; fusidate sodium; fusidic acid;gentamicin sulfate; gloximonam; gramicidin; haloprogin; hetacillin;hetacillin potassium; hexedine; ibafloxacin; imipenem; isoconazole;isepamicin; isoniazid; josamycin; kanamycin sulfate; kitasamycin;levofuraltadone; levopropylcillin potassium; lexithromycin; lincomycin;lincomycin hydrochloride; lomefloxacin; Lomefloxacin hydrochloride;lomefloxacin mesylate; loracarbef; mafenide; meclocycline; meclocyclinesulfosalicylate; megalomicin potassium phosphate; mequidox; meropenem;methacycline; methacycline hydrochloride; methenamine; methenaminehippurate; methenamine mandelate; methicillin sodium; metioprim;metronidazole hydrochloride; metronidazole phosphate; mezlocillin;mezlocillin sodium; minocycline; minocycline hydrochloride; mirincamycinhydrochloride; monensin; monensin sodium; nafcillin sodium; nalidixatesodium; nalidixic acid; natainycin; nebramycin; neomycin palmitate;neomycin sulfate; neomycin undecylenate; netilmicin sulfate;neutramycin; nifuiradene; nifuraldezone; nifuratel; nifuratrone;nifurdazil; nifurimide; nifiupirinol; nifurquinazol; nifurthiazole;nitrocycline; nitrofurantoin; nitromide; norfloxacin; novobiocin sodium;ofloxacin; onnetoprim; oxacillin; oxacillin sodium; oximonam; oximonamsodium; oxolinic acid; oxytetracycline; oxytetracycline calcium;oxytetracycline hydrochloride; paldimycin; parachlorophenol; paulomycin;pefloxacin; pefloxacin mesylate; penamecillin; penicillin G benzathine;penicillin G potassium; penicillin G procaine; penicillin G sodium;penicillin V; penicillin V benzathine; penicillin V hydrabamine;penicillin V potassium; pentizidone sodium; phenyl aminosalicylate;piperacillin sodium; pirbenicillin sodium; piridicillin sodium;pirlimycin hydrochloride; pivampicillin hydrochloride; pivampicillinpamoate; pivampicillin probenate; polymyxin B sulfate; porfiromycin;propikacin; pyrazinamide; pyrithione zinc; quindecamine acetate;quinupristin; racephenicol; ramoplanin; ranimycin; relomycin;repromicin; rifabutin; rifametane; rifamexil; rifamide; rifampin;rifapentine; rifaximin; rolitetracycline; rolitetracycline nitrate;rosaramicin; rosaramicin butyrate; rosaramicin propionate; rosaramicinsodium phosphate; rosaramicin stearate; rosoxacin; roxarsone;roxithromycin; sancycline; sanfetrinem sodium; sarmoxicillin;sarpicillin; scopafungin; sisomicin; sisomicin sulfate; sparfloxacin;spectinomycin hydrochloride; spiramycin; stallimycin hydrochloride;steffimycin; streptomycin sulfate; streptonicozid; sulfabenz;sulfabenzamide; sulfacetamide; sulfacetamide sodium; sulfacytine;sulfadiazine; sulfadiazine sodium; sulfadoxine; sulfalene;sulfamerazine; sulfameter; sulfamethazine; sulfamethizole;sulfamethoxazole; sulfamonomethoxine; sulfamoxole; sulfanilate zinc;sulfanitran; sulfasalazine; sulfasomizole; sulfathiazole; sulfazamet;sulfisoxazole; sulfisoxazole acetyl; sulfisboxazole diolamine;sulfomyxin; sulopenem; sultamricillin; suncillin sodium; talampicillinhydrochloride; teicoplanin; temafloxacin hydrochloride; temocillin;tetracycline; tetracycline hydrochloride; tetracycline phosphatecomplex; tetroxoprim; thiamphenicol; thiphencillin potassium;ticarcillin cresyl sodium; ticarcillin disodium; ticarcillin monosodium;ticlatone; tiodonium chloride; tobramycin; tobramycin sulfate;tosufloxacin; trimethoprim; trimethoprim sulfate; trisulfapyrimidines;troleandomycin; trospectomycin sulfate; tyrothricin; vancomycin;vancomycin hydrochloride; virginiamycin; or zorbamycin.

Further provided herein are methods of inhibiting a bacterialribonuclease, such as the protein component of Staphylococcus aureusRNase P. In some embodiments, the bacterial ribonuclease is RnpA. Themethods comprise contacting the bacterial ribonuclease with an effectiveamount of one or more of the compounds or compositions described herein.Such amounts are sufficient to achieve a therapeutically effectiveconcentration of the compound or active component of the composition invivo or in vitro.

The methods and compounds as described herein are useful for bothprophylactic and therapeutic treatment. As used herein the term treatingor treatment includes prevention; delay in onset; diminution,eradication, or delay in exacerbation of signs or symptoms after onset;and prevention of relapse. For prophylactic use, a therapeuticallyeffective amount of the compounds and compositions or pharmaceuticallyacceptable salts thereof as described herein are administered to asubject prior to onset (e.g., before obvious signs of a bacterialinfection), during early onset (e.g., upon initial signs and symptoms ofa bacterial infection), or after an established inflammatory response ordevelopment of a bacterial infection. Prophylactic administration canoccur for several days to years prior to the manifestation of symptomsof an infection. Prophylactic administration can be used, for example,in the preventative treatment of subjects exposed to Staphylococcusaureus. Therapeutic treatment involves administering to a subject atherapeutically effective amount of the compounds and compositions orpharmaceutically acceptable salts thereof as described herein after abacterial infection is diagnosed.

Kits

Also provided herein are kits for treating or preventing inflammation orcancer in a subject. A kit can include any of the compounds orcompositions described herein. For example, a kit can include a compoundof Formula I. A kit can further include one or more antibacterial agents(e.g., oxacillin). A kit can include an oral formulation of any of thecompounds or compositions described herein. A kit can additionallyinclude directions for use of the kit (e.g., instructions for treating asubject).

The examples below are intended to further illustrate certain aspects ofthe methods and compounds described herein, and are not intended tolimit the scope of the claims.

EXAMPLES

S. aureus RNA degradation factors were empirically identified and, asdemonstrated below, were proven to represent promising antimicrobialdrug development targets. To do so, the fact that S. aureus owes itsability to cause infection, in part, to the temporal expression of anexpansive repertoire of virulence factors, many of which are regulatedin a cell density-dependent manner during laboratory culture conditions,was exploited (see Novick, R. P., Mol Microbiol, 48: 1429-1449 (2003)).Studies were then performed to determine whether growth phase regulatedchanges in S. aureus virulence factor expression occur at the level ofmRNA degradation and whether the proteins involved in this process mayinclude members of the organism's RNA degradation machinery.Accordingly, Affymetrix GeneChips were used to compare the mRNA decayrates of well-characterized S. aureus virulence factors duringexponential- and stationary-phase growth.

Results revealed that the mRNA turnover properties of many S. aureusvirulence factor transcripts differed between the two growth phases.Furthermore, the global mRNA decay properties of exponential andstationary phase cells were found to be dramatically different; 884 S.aureus mRNA species were stabilized during stationary phase growth.Among the genes whose expression correlated with mRNA decay was theprotein component of ribonuclease P, RnpA, suggesting that it may play arole in bulk mRNA turnover. Consistent with that possibility, it wasdemonstrated that recombinant S. aureus RnpA exhibits ribonucleaseactivity in vitro and RnpA depleted cells exhibit reduced mRNAdegradation. Because RnpA is an essential S. aureus enzyme with lowamino acid conservation with mammalian proteins, it is an appropriatetarget for antimicrobial drug discovery. Accordingly, high through-putand secondary screening assays were used to identify small moleculeinhibitors of RnpA-mediated RNA degradation. One of these agents wasshown to inhibit S. aureus mRNA turnover, exhibited antimicrobialactivity against MRSA, VISA, and VRSA, as well as other Gram-positivepathogens with high RnpA conservation, and limited pathogenesis in amurine acute lethal model of infection. Collectively these resultsdemonstrate that RnpA is a previously uncharacterized member of the S.aureus RNA degradation machinery and validate its utility as anantimicrobial drug discovery target.

Example 1 Growth-Phase Dependent Alternations in S. Aureus Turnover

For half life determinations, S. aureus strain UAMS-1, RN4220 (pCN51;plasmid containing CdCl₂ inducible promoter), RN4220 (pRNPA; pCN51capable of producing full length rnpA mRNA), or RN4220 (pRNPA-A.S.;pCN51 capable of producing rnpA antisense RNA) were grown tomid-exponential or stationary phase, transcription was arrested by theaddition of rifampin (200 μg/ml), and aliquots were removed at 0-, 2.5-,5-, 15- and 30-min post-transcriptional arrest for strain UAMS-1. Toconserve reagents, aliquots were removed at 0 and 10 minpost-transcriptional arrest for RN4220 derivatives. Plating ensuredcultures had not developed rifampin resistance. Each strain and/orgrowth phase was assessed twice, except for RN4220 pRNPA-A.S. cellswhich were assessed four times. RNA was isolated from each aliquot,labeled, hybridized to an S. aureus GeneChip (Affymetrix; Santa Clara,Calif.), duplicates were averaged, and the mRNA half-lives of all mRNAspecies were determined, as previously described (see Anderson et al., JBacteriol, 188: 6739-6756 (2006); Roberts et al., J Bacteriol, 188:2593-2603 (2006)). To measure the mRNA turnover characteristics ofRNPA1000 challenged cells, exponential-phase S. aureus were treated with0.5× MIC of the RnpA inhibitor or equivalent volume compound solvent(DMSO) for 30 min. Transcript synthesis was then arrested and thetranscript titers of mRNA species were measured at 0- and 5-minpost-transcriptional arrest (see Anderson et al., J Bacteriol, 188:6739-6756 (2006); Roberts et al., J Bacteriol, 188: 2593-2603 (2006)).

The results demonstrated that the mRNA turnover properties of many (41%)virulence factor transcripts differed between the two growth phases,suggesting that regulated changes in mRNA turnover may affect theirexpression. Moreover, it was observed that the organism produced atleast five stationary phase specific small stable RNAs (SSRs), ahypothesized class of regulatory non-coding RNA molecules (see Andersonet al., J Bacteriol, 188: 6739-6756 (2006); Roberts et al., J Bacteriol,188: 2593-2603 (2006)). Further, the global mRNA turnover properties ofexponential- and stationary-phase cells differed considerably.Consistent with previous measurements, it was found that most (90%)exponential phase transcripts are rapidly degraded (half life of ≦5min), 9% exhibit intermediate stability (half life of >5 min but ≦30min), and 1% are stable (half life of >30 min) (see Anderson et al., JBacteriol, 188: 6739-6756 (2006); Roberts et al., J Bacteriol, 188:2593-2603 (2006)). However, during stationary phase growth, 76%, 21%,and 3% of mRNA species exhibit short, intermediate, and stable halflives, respectively (FIG. 1). Neither RNase J1 nor RNase Y were found tobe differentially expressed in a growth phase dependent manner. Amongthe 367 genes repressed during stationary phase growth was rnpA, whichcodes for the protein component of ribonuclease P.

Example 2 S. Aureus RnpA Exhibits Ribonuclease Activity and AffectsCellular mRNA Degration

Protein Purification

Each putative S. aureus ribonuclease predicted open reading frame wasPCR amplified and inserted into the ligation-independent cloning site ofplasmid pET-30 Ek/LIC (Novagen; Madison Wis.). Sequencing confirmed thatthis fused a hexahistidine-tag to the N-terminus of each protein underthe control of the plasmid's isopropyl β-D-1-thiogalactopyranoside(IPTG) inducible promoter. Following transformation, each protein waspurified from E. coli BL21 (DE3) cells grown in the presence of IPTG (4hr) by Ni⁺² affinity chromatography. More specifically, 10 g of cellpellet was suspended in 50 ml of buffer A (300 mM NaCl, 50 mM Na₂HPO₄,pH 7.4) containing a complete mini EDTA-free protease inhibitor tablet(Roche; Branford, Conn.) and 20 mM imidazole. Cells were ruptured byseven passes at 15,000 psi through an Emulsifex-C3 microfluidizer(Avestin Inc.; Ottawa, Canada). Cell debris was removed bycentrifugation at 12,000×g for 30 min and supernatants were loaded ontoa 5 mL Ni-NTA FF-crude affinity column (GE Healthcare Bio-Sciences;Piscataway, N.J.) with an AKTA-FPLC high performance liquidchromatography system (GE Healthcare Bio-Sciences; Pittsburgh, Pa.).Proteins eluted in a single peak with a linear imidazole gradient (80 mMto 500 mM) in buffer A. The presence of each protein was assessed byCoomasie stained SDS-PAGE and matrix-assisted laserdesportion/ionization (MALDI) analysis spectrometry (Wistar Institute;Philadelphia, Pa.).

Plasmids

Plasmids pRNPA-S and pRNPA-A.S. contain the putative rnpAtranscriptional unit including predicted Shine-Delgarno sequence in thesense and antisense orientation, respectively under control of the CdCl₂inducible of the S. aureus shuttle-vector pCN51 (see Charpentier et al.,Appl Environ Microbiol, 70: 6076-6085 (2004)). Briefly, the rnpA openreading frame and 34 nt upstream sequence was PCR amplified from S.aureus strain UAMS-1 using primers 5′GAATTCTCAAATAAAAACGATAAATAAGCGAGTGATGTTA (forward) (SEQ ID NO. 8) and 5′GGTACCTTACTTAATCTTTTTATTAAAAACTTTGGCAA (reverse) (SEQ ID NO. 9)containing a 5′ terminal EcoRI and KpnI restriction enzyme site(underlined), respectively, or primers in which the restriction enzymesequence had been reversed. Resulting PCR products were ligated intopCRII-TOPO vector and transformed into E. coli INVαF′ cells forpropagation (Invitrogen, Carlsbad, Calif.). Plasmid DNA was subsequentlypurified using QIAprep Spin Miniprep Kits (Qiagen, Valencia, Calif.)then digested with EcoRI and KpnI to liberate the plasmid inserts, whichwere gel purified using a QIAquick Gel Extraction Kit (Qiagen) andligated into EcoRI and KpnI-digested pCN51. DNA sequencing confirmed theintegrity of plasmid pRNPA-S and pRNPA-A.S.

Western Blotting

Affinity purified PolyQuik rabbit S. aureus RnpA polyclonal antibodieswere generated by Invitrogen (Carlsbad, Calif.). Total bacterialproteins were isolated from RN4220 cells containing plasmid vector(pCN51), RnpA overexpressor plasmid (pRNPA-S) or RnpA antisense RNAplasmid (pRNPA-A.S.) following 30 min growth in TSB medium supplementedwith 2.5 μM CdCl₂ to induce RNA expression and 10 μg/ml erythromycin forplasmid maintenance. Resultant protein concentrations were determined byconventional Bradford Assays and 2.0 μg of each protein sample orpurified S. aureus RnpA was electrophoresed in a 10% SDS polyacrylamidegel and transferred to a polyvinylidene fluoride membrane (Millipore,Billerica, Mass.). Membranes were blocked with 10% milk, washed,incubated with rabbit RnpA antibody (1:1000 dilution), washed, incubatedwith horseradish peroxidase-conjugated anti-rabbit antibody (1:1000dilution; GE Healthcare) and processed using an Amersham ECL WesternBlotting System, according to the manufacturer's recommendations (GEHealthcare).

Results

Recombinant S. aureus RnpA was found to catalyze digestion of rRNA andstaphylococcal protein A (spa) mRNA (FIGS. 2B and 2C), as well as threeother mRNA species tested. Other putative S. aureus ribonucleasesincluding RNase III, RNase HII, RNase HIII, RNase Y, RNase J1, and BNdid not exhibit equivalent RNA degradation activity during these assayconditions (FIG. 2B). SDS-PAGE and matrix-assisted laserdesorption/ionization (MALDI) analysis confirmed that the observedribonuclease activity was associated with the presence of S. aureus RnpA(FIG. 2A). In FIG. 2A, the band at about 17.2 kDa (solid arrow; Band 2)was confirmed to be S. aureus RnpA by tandem mass spectrometry (WistarInstitute; Philadelphia, Pa.), whereas top-hits for minor contaminants(dashed arrows) were determined to be E. coli 50S ribosomal protein L3(Band 1) or S. aureus RnpA polypeptide fragments, corresponding to aminoacids 11-107 (Bands 3 and 4) or 12-107 (Band 5). Nonetheless, SDS-PAGEassessment of approximately 1000-fold excess (25 μg) of RnpApurification product used in the aforementioned ribonuclease assaysrevealed trace amounts of four additional polypeptides within theprotein preparation, raising the possibility that contaminating E. coliribonucleases may be present with the RnpA product. MALDI analysisrevealed the identity of these proteins to be E. coli ribosomal proteinL3, and three S. aureus RnpA fragments, presumably reflectingproteolytic degradation of full length RnpA during protein preparationas opposed to mature alternative translation products. No E. coliribonucleases were detected, suggesting that the protein preparation'sribonucleolytic activity could be attributed to S. aureus RnpA.Moreover, reverse transcriptase mediated PCR revealed that E. coli rnpBwas undetectable within the preparation, establishing that RnpAribonuclease activity was not due to the formation of chimeric RNase Pmolecules consisting of S. aureus RnpA and E. coli rnpB RNA. Indeed, invitro synthesized E. coli rnpB neither catalyzed S. aureus RNAdegradation (alone) nor affected the activity of RnpA-mediated RNAdigestion during both standard and elevated Mg⁺² reaction conditions.

While S. aureus RNase J1 exhibited low ribonucleolytic activity in thereaction conditions used here, subsequent studies revealed that it is apotent ribonuclease in differing buffering conditions (see Even et al.,Nucleic Acids Res, 33: 2141-2152 (2005)) and could be used as a controlto further evaluate the putative in vitro ribonuclease activity of S.aureus RnpA. More specifically, it was assessed whether RnpA-mediatedspa mRNA degradation could be inhibited by the addition of affinitypurified rabbit polyclonal S. aureus RnpA antibodies. Initial studiesdid not reveal that antibody limited either RnpA or RNase J1ribonucleolytic activity. However, anticipating that the only a subsetof antibodies within the immunoglobulin mixture may recognize RnpAepitope(s) that affect the enzyme's activity, reverse transcription PCRamplification of spa-digested products was used as a more sensitivemeans of monitoring what, if any, effect the antibody had onRnpA-mediated transcript degradation. Results revealed that antibodyaddition did indeed weakly inhibit RnpA-mediated degradation of fulllength spa mRNA but had no effect on RNase J1 activity (FIG. 2D).Equivalent amounts of pre-immune serum had no effect on RnpA activity.Taken together, these data suggest that a previously unrecognizedfunction S. aureus RnpA is that of RNA digestion.

Small molecule inhibitors of essential bacterial RNA turnover proteinsare expected to interfere with bacterial growth and represent a newclass of antimicrobial agents. In that regard, S. aureus RnpA is areported essential enzyme (see Chaudhuri et al., BMC Genomics, 10: 291(2009); Ji et al., Science, 293: 2266-2269 (2001)) and thus could beconsidered a target for chemotherapeutic development. Indeed, inductionof an antisense RNA molecule that is predicted to be complementary tothe −34 to +353 rnpA mRNA translation start site (under control of thecadmium chloride inducible promoter of plasmid, pCN51 (see Charpentieret al., Appl Environ Microbiol, 70: 6076-6085 (2004)) limited S. aureusproliferation in the presence of 10 μM inducer. Conversely, no growthdefects were observed for cells expressing the corresponding sensestrand RNA molecule or the antisense plasmid strain in the absence ofinducer (FIG. 6). These results indicate that S. aureus RnpA is anessential protein. Further, using this rnpA antisense RNA system, it wasassessed whether RnpA affects S. aureus cellular mRNA turnover.Accordingly, the RNA degradation properties were measured for cellsharboring plasmid vector alone or cells containing plasmid borne copiesof rnpA mRNA or rnpA antisense RNA during growth in the presence of 2.5μM CdCl₂. As shown in FIG. 6, 2.5 μM cadmium chloride was empiricallydetermined to be the optimal concentration that allowed increased- ordecreased-RnpA production within rnpA mRNA or rnpA antisense expressingstrains, respectively, but did not limit bacterial growth of theantisense RNA producing strain. Accordingly, RNA turnover analysesrevealed that diminished RnpA levels correlated with the stabilizationof many mRNA species, suggesting that the enzyme contributes to bulkcellular RNA degradation. More specifically, it was found that 88% and87% of all exponential phase transcripts produced in RnpA overexpressingand vector containing cells exhibited a half life of less than 10 min,respectively. The finding that RnpA overexpression did not acceleratecellular RNA degradation may indicate that the protein's RNA degradationactivity is dependent on co-factors, which remain at wild type levels orthat the protein did not reach a concentration that effectivelyincreases RNA turnover. Regardless, 63% of transcripts produced in RnpAdepleted cells exhibited a half life of less than 10 min, suggestingthat the protein contributes to S. aureus mRNA turnover (FIG. 5A).

Example 3 Identification of Small Molecule Inhibitors of RnpA-MediatedRNA Degradation

The above results indicate that S. aureus RnpA is an essential enzymethat exhibits in vitro ribonuclease activity and participates incellular RNA degradation. Moreover, the protein is well conserved acrossGram-positive bacteria but lacks amino acid conservation with mammalianproteins, making it an attractive target for novel antibiotic drugdevelopment. Accordingly, a fluorescence-based high through-put assaywas used to screen 29,066 commercial compounds (ActiProbe-25K andNatural product libraries; Timtec; Newark, Del.) for small moleculeinhibitors of RnpA-mediated RNA degradation (FIG. 3A).

Specifically, members of the ActiProbe-25K and Natural Product libraries(29,940 compounds total; TimTec Inc.; Newark, Del.) were screened forsmall molecule inhibitors of S. aureus RnpA mediated total bacterial RNAdegradation. All reactions (50 μl) were performed in 96-well format andcontained 20 pmol RnpA, 200 ng S. aureus total RNA, and about 5 μM ofeach compound in 1× reaction buffer (2 mM NaCl, 2 mM MgCl₂, 50 mMTris-HCl, pH 6.0). Mixtures were incubated at 37° C. for 20 min at whichtime Quant-iT RiboGreen® (100 μl; Invitrogen) was added to quantify theamount of RNA substrate remaining. Percent enzyme inhibition wascalculated as remaining substrate/starting substrate*100. For inhibitorytitration assays, 1 pmol of spa mRNA was incubated with 20 pmol RnpAalone (positive control) or in the presence of increasing amounts (0,25, 50, 100, 125, 150, 200, 250, and 500 μM) RNP1000 for one hour at 37°C. Following this, 20 μl of each reaction mixture were subjected toelectrophoresis in a 1.2% formaldehyde-containing agarose gel andvisualized by ethidium bromide staining.

In total, fourteen molecules inhibited the enzyme's RNA turnoveractivity by ≧50%. A gel-based secondary assay confirmed that five ofthese molecules were bona-fide inhibitors of RnpA-mediated RNAdegradation (FIG. 3B). One of these compounds, RNPA1000 (FIG. 3C;IC₅₀=100-125 μM), did not affect the activity of the commerciallyavailable E. coli RNase HI, RNase A, RNase I or in-house purified S.aureus RNase J1 at any concentration tested (0-750 μM), but did mildlyinhibit E. coli RNase III activity (IC₅₀=500-750 μM; data not shown).These and other data (see below) suggest that RNPA1000 may havespecificity for S. aureus RnpA, yet as with any small molecule we cannotrule out the possibility that the agent may also affect other S. aureusenzymes. To assess whether RnpA-inhibitory agents exhibit potential asantimicrobials, a series of experiments were performed to evaluatewhether RNPA1000 inhibited S. aureus growth and could limit S. aureuspathogenesis in a systemic model of infection.

Example 4 Antimicrobial Susceptibility Testing

With the exception of RN4220-derivatives, in vitro activities ofRNPA1000 against bacteria were determined by the broth microdilutionmethod according to the Clinical and Laboratory Standards Institute(CLSI) guidelines using cation adjusted Mueller-Hinton broth or MH brothsupplemented with 5% lysed horse blood (for testing Streptotococcusspp.). Microtiter plates containing serial dilutions of RNPA1000 (0, 4,8, 16, 32, 64, and 128 μg/ml) were inoculated with 10⁵ colony formingunits (CFU)/ml and incubated for 18 hr at 37° C. The MIC for eachisolate was defined as the lowest concentration of RNPA1000 thatcompletely inhibited growth of the organism as detected by the unaidedeye. The MIC for each S. aureus strain was further refined by repeattesting following the procedure described above, except that microtiterwells contained 1 μg/ml incremental increases in concentration ofRNPA1000 spanning the lowest concentration that initially did notcompletely inhibit growth (16 μg/ml) and the concentration thatcompletely inhibited growth (32 μg/ml). The MIC value for each S. aureusstrain was determined to be the median score of replicate measurements(n=5). Wells containing concentrations of RNPA1000≧MIC were plated forminimal bactericidal measurement. Where possible, experiments with VRSAstrains were performed in a laminar flow hood to minimize potential forequipment contamination. For RN4220 cells containing plasmid vector(pCN51), RnpA overproducing plasmid (pRNPA-S) or RnpA underproducingplasmid (pRNPA-A.S.) in vitro antimicrobial activity of RNPA1000 wasperformed by the microdilution method as described above, except thatcells were grown in Tryptic Soy Broth medium supplemented with 2.5 μMCdCl₂ and 0, 1, 2, 4, 8, 16, 32, 64, or 128 μg/ml RNPA1000. Time-killassays were also performed to monitor the antimicrobial properties ofRNPA1000 for S. aureus strain UAMS-1 in the absence and presence of0.25, 0.5, 2, and 4 times the strain's MIC for oxacillin (1 μg/ml),rifampicin (0.5 μg/ml), vancomycin (2 μg/ml), or daptomycin (1 μg/ml).The indicated amount of RNPA1000 and/or commercial antibiotic were addedto mid-exponential phase (2×10⁸ cfu/ml) S. aureus strain UAMS-1 cellsand incubated at 37° C. Aliquots were removed at 0, 2, 4, 8, and 24 hrpost-antimicrobial challenge, serial diluted, and plated to enumerateresulting cfu/ml. All time-kill assays were repeated at least 3 times.Results are provided in Table 1.

TABLE 1 Organism (Phenotype) Strain^(b) MIC (μg/ml)^(b, c) Organism(Phenotype) Strain^(b) MIC (μg/ml)^(c, d) S. aureus (MRSA) USA100 26 S.pneumoniae (MDR) Isolate 4 32 S. aureus (MRSA) USA200 32 S. pneumoniae(MDR) Isolate 5 16 S. aureus (MRSA) USA300 23 S. pyogenes Isolate 1 8 S.aureus (MRSA) USA400 23 S. sanguis Isolate 1 16 S. aureus (MRSA) USA50023 S. bovis ATCC49147 32 S. aureus (MRSA) USA600 32 E. faecalis Isolate1 64 S. aureus (MRSA) USA700 32 E. faecalis Isolate 2 64 S. aureus(MRSA) USA800 23 E. faecalis Isolate 3 64 S. aureus (MRSA) USA900 32 E.faecalis Isolate 4 64 S. aureus (MRSA) USA1000 29 E. faecalis Isolate 564 S. aureus (MRSA) USA1100 32 E. faecium Isolate 1 64 S. aureus (MSSA)UAMS-1 26 E. faecium Isolate 2 64 S. aureus (VISA) VISA-NRS1 32 E.faecium Isolate 3 64 S. aureus (VISA) VISA-NRS3 16 E. faecium Isolate 464 S. aureus (VISA) Isolate 3 16 E. faecium Isolate 5 64 S. aureus(VISA) Isolate 4 32 E. faecium Isolate 5 + 32 S. aureus (VISA) Isolate 516 reserpine S. aureus (VRSA) VRSA-VRS1 16 E. faecium (VRE) Isolate 1 64S. aureus (VRSA) VRSA-VRS10 32 E. faecium (VRE) Isolate 2 64 S.epidermidis Isolate 1 16 E. faecium (VRE) Isolate 3 32 S. epidermidisIsolate 2 8 E. faecium (VRE) Isolate 4 64 S. epidermidis Isolate 3 8 E.faecium (VRE) Isolate 5 32 S. epidermidis Isolate 4 8 B. cereus Isolate1 8 S. epidermidis Isolate 5 8 E. coli Isolate 1 >64 S. agalactiaeIsolate 1 16 E. coli Isolate 2 >64 S. agalactiae Isolate 2 32 E. coliIsolate 3 >64 S. agalactiae Isolate 3 32 E. coli Isolate 4 >64 S.agalactiae Isolate 4 32 E. coli Isolate 5 >64 S. pneumoniae Isolate 1 16A. baumannii Isolate 1 >64 S. pneumoniae Isolate 2 16 A. baumanniiIsolate 2 >64 S. pneumoniae Isolate 3 16 A. baumannii Isolate 3 >64 S.pneumoniae Isolate 4 32 A. baumannii Isolate 4 >64 S. pneumoniae Isolate5 16 A. baumannii Isolate 5 >64 S. pneumoniae (MDR) Isolate 1 32 A.baumannii Isolate 5 + >64 S. pneumoniae (MDR) Isolate 2 32 reserpine S.pneumoniae (MDR) Isolate 3 16 ^(a)Organism and relavent antibioticresistance phenotype provided (in parenttheses); methicillin resistantS. aureus (MRSA); vancomycin intermediate S. aureus (VISA); vancomycinresistant S. aureus (VRSA); multidrug resistant S. pneumoniae (MDR);vancomycin resistant E. faecium (VRE). ^(b)With the exception of S.aureus and S. bovis, all strains were clinical blood isolates; USA-types(U.S. MRSA lineages) were obtained from the Centers for Disease Controland Prevention; S. aureus strain UAMS-1 is a clinical osteomyelitisisolate; isolates NRS1, NRS3, VRS1 and VRS10 were obtained through theNetwork of Animicrobial Resistance in Staphylococcus aureus (NARSA) ; S.bovis strain ATCC49147 was obtained from the American Type CultureCollection. ^(c)Each S. aureus isolate was tested 5 times; otherorganisms were tested in duplicate. ^(d)Minimum inhibitory concentration(MIC) was determined following the Clinical and Laboratory StandardsInstitute (CLSI) guidelines for antimicrobial susceptibility testing. S.aureus MRSA isolates were subsequently more accurately measured.

As shown in Table 1, RNPA1000 demonstrated moderate antimicrobialactivity against two well-characterized genotypically diverse S. aureusisolates, UAMS-1 (clinical osteomyelitis isolate; MIC 26 μg/ml) andUSA300-0114 [predominant cause of U.S. community-associated methicillinresistant S. aureus infections (MRSA); MIC 23 μg/ml], as well asrepresentatives of other major MRSA lineages circulating throughout theUS (see McDougal et al., J Clin Microbiol, 41: 5113-5120 (2003)).Likewise, RNPA1000 demonstrated antimicrobial activity againstvancomycin-intermediate susceptible S. aureus (VISA) and vancomycinresistant S. aureus (VRSA). Time kill assays revealed that RNPA1000 actsas a bacteriostatic agent (FIG. 7, Panel A), and that it does not affectthe antimicrobial activities of other anti-staphylococcal agents,including vancomycin, daptomycin, or rifampicin (data not shown), butdoes mildly increase the potency of oxacillin (FIG. 7, Panels B and C).The RnpA-inhibitor also exhibited antimicrobial activity againstStaphylococcus epidermidis, antibiotic susceptible and multi-drugresistant Streptococcus pneumoniae, Streptococcus pyogenes,Streptococcus agalactiae, and Bacillus cereus. RNPA1000 also showed mildactivity against Enterococcus faecalis, Enterococcus faecium andvancomycin resistant E. faecium (VRE), but did not affect Escherichiacoli or Acinetobacter baumannii growth (Table 1). The latter wasexpected because E. coli and A. baumannii RnpA share limited amino acididentity (24% and 26%, respectively) with S. aureus RnpA (FIG. 8).Moreover, purified A. baumannii RnpA did not demonstrate ribonuclolyticactivity in our assay conditions (data not shown). Enterococcisusceptibility to RNPA1000 was increased from an MIC of 64 μg/ml to 32μg/ml in the presence of the efflux pump inhibitor reserpine, suggestingthat enterococci may be inherently susceptible to the RnpA inhibitor.Conversely, the efflux inhibitor had no effect on A. baumannii RNPA1000susceptibility (Table 1). Taken together, these results indicate thatbacterial RNPA1000 susceptibility correlates with amino acid similarityto S. aureus RnpA and the enzyme's RNA degradation activity.

To assess whether the susceptibility of S. aureus to RNPA1000 wasattributable to the inhibition of cellular RnpA, the mRNA turnoverproperties of S. aureus that were challenged with a sub-inhibitoryconcentration of RnpA-inhibitor (0.5× MIC) were directly measured.Following 30 min treatment, RNPA1000 reduced the mRNA degradation rateof S. aureus cells, in comparison to mock treated cells (FIG. 5A). Thus,RnpA-inhibitory compounds reduce cellular mRNA degradation, presumablyby limiting the enzyme's cellular function. The mRNA turnover propertiesof RNPA1000 treated cells resembled that of RnpA depleted cells (FIG.2E), suggesting that the agent may be affecting the enzyme. To moredirectly determine whether RNPA1000's antimicrobial effects are mediatedthrough cellular inhibition of RnpA, the RNPA1000 susceptibility of S.aureus RnpA over- and under-producing cells was assessed. S. aureusharboring vector, or a plasmid copy of wild type rnpA mRNA or rnpAantisense RNA under control of the CdCl₂ inducible promoter were grownin the presence of 2.5 μM inducer and increasing concentrations ofRNPA1000. As stated above, this concentration of cadmium chlorideinduces mild changes in RnpA protein expression (RnpA overproduction orunderproduction) but is modest enough that cellular growth is notaffected. As shown in FIG. 5B, both vector containing- and RnpAoverproducing-cells exhibited an MIC of 32 μg ml⁻¹, whereas the MIC ofRnpA underproducing cells was 8 μg ml⁻¹. The latter indicates that S.aureus' RNPA1000 susceptibility correlates to cellular RnpA levels andthat the agent's antimicrobial mode-of-action is, in part, RnpAdependent.

Example 5 Cytotoxicity Assays

It was then assessed whether RnpA-inhibitory agent concentrationscorresponding to the effective bacterial MIC values (10-50 μg/ml)elicited human cell cytotoxicity. HepG2 human hepatocytes (10⁵ cells)were seeded in individual wells of a microtitre plate and incubated for16 hr at 37° C. with 5% carbon dioxide in Dulbecco's Modified EagleMedia supplemented with 10% fetal bovine serum. Cells were thenchallenged with Mitomycin C (5 μg/ml; positive control) or 0, 25, or 50μg/ml RNPA1000 for either 24 or 48 hrs. Cell viability was measuredspectrophotometrically (570 nm) following the addition and subsequentreduction of tetrazolium salt (MTT) within metabolically active cells,as per the manufacturer's recommendations (American Type CultureCollection; Manassas, Va.).

MTT cell proliferation assay measurements revealed that 24 hrRnpA-inhibitor exposure did not cause human HepG2 cell toxicity at anyconcentration tested (data not shown). However, extended RNP1000exposure (48 hr) elicited mild cytotoxicity at 25 μg/ml, whichcorresponds to the minimum inhibitory concentration of most MRSAlineages (FIG. 4A), whereas higher concentrations exhibited increasedtoxicity (data not shown).

Example 6 Antimicrobial Efficacy of RnpA-Inhibitor on Biofilm-AssociatedBacteria

The success of S. aureus as a bacterial pathogen can be attributable, inpart, to its ability to form biofilms on implanted medical devices,which presumably provides a focus for bacterial dissemination tosecondary host sites. One of the complicating issues in treatingbiofilm-associated infections is that biofilm-associated bacteria areinherently recalcitrant to antibiotic treatment. For instance, onerecent in vitro study showed that despite using a strain that wasintrinsically susceptible to each antibiotic, 5× MIC of daptomycin,linezolid, or vancomycin only reduced biofilm-associated bacteria by <2logs following 24 hr treatment and none of these antibiotics clearedbiofilm-associated S. aureus even when administered at 20× MIC over acourse of 3 days (see Weiss et al., Antimicrob Agents Chemother, 53:2475-2482 (2009)). Transcription profiling studies have revealed thatdespite being physiologically unique, biofilm-associated S. aureusresemble planktonic stationary phase cells (see Beenken et al., JBacteriol, 186: 4665-4684 (2004)). Indeed, similar to stationary phasebacteria, rnpA expression is diminished 4.3 and 6.2-fold in S. aureusbiofilm-associated and biofilm-detached bacteria, respectively, incomparison to exponential phase cells (Dunman and Horswill,unpublished). Because low levels of RnpA are likely to be present withinbiofilm-associated bacteria, fewer RnpA-inhibitory molecules could berequired to interfere with the protein's function and, consequently,antimicrobial activity. Thus, biofilm-associated S. aureus may exhibitconsiderable susceptibility to an RnpA-inhibitor, such as RNPA1000.

To determine this, in vitro biofilm assays were performed as describedin Weiss et al., Antimicrob Agents Chemother, 53: 2475-2482 (2009).Briefly, 1 cm segments of 14-gauge fluorinated ethylene propyleneIntrocan Safety Catheters (B. Braun, Bethlehem, Pa.) were coated withhuman plasma and placed in individual wells of a 12-well microtiterplate containing 2 ml biofilm medium and S. aureus strain UAMS-1 at afinal OD_(600 nm) of 0.05. Following overnight incubation at 37° C.catheters were removed, rinsed in phosphate buffered saline (PBS), andtransferred to fresh biofilm medium containing 0, 5, 10, or 20 times theS. aureus MIC for RNPA1000. Catheters exposed to each dose (n=3) wererecovered daily over a period of 3 days, with the medium being replacedeach day. After each recovery time point catheters were rinsed in PBSand adherent bacteria were enumerated by sonication and plating.Analysis of variance (ANOVA) of logarithmically-transformed bacterialcount data was used to evaluate the effect of RNPA1000 exposure.

As shown in FIG. 4C, treatment of biofilm-associated S. aureus with 5×MIC RNPA1000 for 24 hr resulted in a 3-log decrease in bacterial burden,suggesting that during short term exposure the agent is equally, if notmore potent, than daptomycin, vancomycin, or linezolid. Further, whilebacterial clearance was never achieved, increasing the length ofexposure or RNPA1000 concentration enhanced antimicrobial activity.Maximal RNPA1000 antimicrobial potency (5-log reduction inbiofilm-associated bacteria) compared favorably with the activities ofcommercially available antibiotics assessed in the same model andconditions (6-log decrease daptomycin, 5-log decrease linezolid; 4-logdecrease vancomycin) (see Weiss et al., Antimicrob Agents Chemother, 53:2475-2482 (2009)). Taken together, these results suggest that RnpA playsan important biological role in S. aureus biofilm maintenance, and thatcorresponding inhibitors may have expanded therapeutic utility intreating biofilm-associated infections.

Example 7 Acute Lethal Model of Infection

Because RNPA1000 was not toxic during short- and only mildly toxicduring extended-HepG2 exposure, it could serve as an appropriate tool toassess whether RnpA-inhibitory molecules are efficacious in a systemicmouse infection model. Female 5-6 week old CD-1 mice were challenged byintraperitoneal injection (0.5 ml) of wild type S. aureus strain Smith,resulting in a final inoculum of 4.55×10⁵ colony forming units/animal;equivalent to 10-100 LD₅₀s and resulted in death of non-treated controlanimals (N=5) within 24 hr post-inoculum. RNPA1000 was solubilized in1:1 mixture of DMSO and PEG400; Vancomycin was prepared in water.Animals (5/dose group) were administered 16, 64, and 256 mg/kg or 0.25,1, 4, and 16 mg/kg of RNPA1000 or Vancomycin, respectively, at 30 minpost infection by subcutaneous injection (0.2 ml). The percent survivinganimals receiving no treatment, a single dose of Vancomycin, orRnpA-inhibitor was recorded daily over the course of the study (5 days).The results are shown in Table 2 and FIG. 4B.

TABLE 2 % Survival Dose (mg/kg) RNPA1000 Vancomycin RNPA1000 (alone) 0 00 100(2) 0.25 — 0 — 1 — 100 — 4 — 100 — 16 20; 20(2) 100 100(2) 64 40;20(2) — 100(2) 256 60; 40; 60(3) — 100(2)

Percent survival refers to a consensus of surviving animals following 5days post-intraperitoneal S. aureus injection and RNPA1000administration. The number in parentheses indicates the number of timesthe experiment was repeated. Each member of non-treated control miceexpired within 24 hr bacterial inoculation (0 mg/kg). Vancomycin servedas a positive control.

As shown in FIG. 4B, subcutaneous injection of RNPA1000 limited thelethal effects of wild type S. aureus injected (4.55×10⁵ cfu/animal)into the intraperitoneal cavity of CD-1 mice. Although this bacterialinoculum (equivalent to 10-100 LD₅₀s) resulted in 100% death ofnon-treated control animals within 24 hr, RNPA1000 provided protectionin a dose-dependent manner. Administration of the highest RnpA-inhibitordose (256 mg/kg) reproducibly resulted in 50% survival, whereas 128mg/kg and 64 mg/kg resulted in 30% and 20% survival, respectively, overthe course of study (FIG. 4B; Table 2). Notably, dosing regimens ofcompound (alone) did not affect animal survival at any of theconcentrations tested (32 mg/kg, 64 mg/kg, 128 mg/kg, 256 mg/kg; Table2). Taken together, these results suggest that RNPA1000 limits bacterialpathogenicity within the acute lethal model of S. aureus infection witha median effective dose (ED₅₀) between 64-256 mg/kg. Thus, RNPA1000could be considered a platform for medicinal chemistry-based generationof more potent derivatives. These results also provide proof of conceptthat RnpA inhibitory agents are efficacious in a systemic mouseinfection model and that RNPA1000 represents a tool to study thecontribution of RnpA to infection processes.

The compounds and methods of the appended claims are not limited inscope by the specific compounds and methods described herein, which areintended as illustrations of a few aspects of the claims and anycompounds and methods that are functionally equivalent are within thescope of this disclosure. Various modifications of the compounds andmethods in addition to those shown and described herein are intended tofall within the scope of the appended claims. Further, while onlycertain representative compounds, methods, and aspects of thesecompounds and methods are specifically described, other compounds andmethods and combinations of various features of the compounds andmethods are intended to fall within the scope of the appended claims,even if not specifically recited. Thus a combination of steps, elements,components, or constituents can be explicitly mentioned herein; however,all other combinations of steps, elements, components, and constituentsare included, even though not explicitly stated.

What is claimed is:
 1. A method of treating or preventing a microbialinfection in a subject, comprising administering to the subject aneffective amount of an RNase inhibitor of the following structure:

or a pharmaceutically acceptable salt or prodrug thereof.
 2. The methodof claim 1, wherein the microbial infection is a bacterial infection. 3.The method of claim 2, wherein the bacterial infection is a Grampositive bacterial infection.
 4. The method of claim 2, wherein thebacterial infection is a Staphylococcus infection.
 5. The method ofclaim 2, wherein the bacterial infection is a Staphylococcus aureusinfection.
 6. The method of claim 5, wherein the Staphylococcus aureusinfection is a drug-resistant Staphylococcus aureus infection.
 7. Themethod of claim 5, wherein the Staphylococcus aureus infection is abiofilm-associated Staphylococcus aureus infection.
 8. The method ofclaim 1, wherein the RNase inhibitor is an RnpA inhibitor.
 9. The methodof claim 1, further comprising administering a second compound, whereinthe second compound is an antibacterial compound.